Biomedical subjects

Toshio Yanagida

Publications and source records attributed to Toshio Yanagida.

At least 37 records · Page 2Linked to original sources

PubMed2006

Optical coherence tomography reveals in vivo cortical plasticity of adult mice in response to peripheral neuropathic pain.

We examined neural plasticity in mice in vivo using optical coherence tomography (OCT) of primary somatosensory (S1) and motor (M1) cortices of mice under the influence of sciatic nerve chronic constriction injury (CCI), a model of neuropathic pain widely utilized in rats. The OCT system used in this study provided cross-sectional images of the cortical tissue of mice up to a depth of about 1mm with longitudinal resolution up to 11 microm. This is the first study to evaluate neural plasticity in vivo using OCT. CCI mice exhibited cold allodynia and spontaneous pain behaviors, which are signs of neuropathic pain, 30 days after sciatic nerve ligation, when OCT observation of S1 and M1 cortices was carried out. The scattering intensity of near-infrared light within the hind paw area of S1 and M1 regions in the contralateral hemisphere was significantly higher than in the ipsilateral hemisphere. These CCI-induced increases in scattering intensity within cortical regions associated with the hind paw probably reflect elevated neural activity associated with neuropathic pain. Synapses and mitochondria are believed to have high light scattering coefficients, since they contain remarkably high concentrations of proteins and complicated membrane structure. Number densities of mitochondria and synapses are known to increase in parallel with increases in neural activity. Our findings thus suggest that neuropathic pain gives rise to neural plasticity within the hind paw area of S1 and M1 contralateral to the ligated sciatic nerve.

Animals↗

PubMed2006

Single supramolecule spectroscopy of natural and alkaline-treated chlorosomes from green sulfur photosynthetic bacteria.

Fluorescence emission properties of intact and alkaline-treated chlorosomes containing bacteriochlorophyll(BChl)-c, d, and e, which were isolated from four species of green sulfur photosynthetic bacteria, were successfully studied at the single-unit level using a total internal reflection fluorescence microscope. Single intact chlorosomes containing BChl-c from Chlorobium (Chl.) tepidum exhibited heterogeneous emission bands of BChl-c self-aggregates. In contrast, fluorescence spectra of chlorosomal BChl self-aggregates in single intact chlorosomes from the other three Chlorobium species were less heterogeneous than those from Chi. tepidum. Removal of energy-accepting BChl-a/protein complexes called baseplates from the intact chlorosomes by treatments with alkaline media hardly changed spectral shapes of BChl aggregates and their peak distributions at the single-chlorosome level. The similarity of spectral properties at the single-unit level between intact and alkaline-treated chlorosomes of four Chlorobium species clearly indicated that the removal of base-plates from intact chlorosomes by the alkaline-treatment did not affect BChl self-aggregates inside single chlorosomes.

Alkalies↗

PubMed2006

Velocity profiles in the rat cerebral microvessels measured by optical coherence tomography.

In order to analyze cerebral hemodynamics and its change following neural activation, the cross-sectional profiles of blood flow velocity in the rat pial microvessels and their temporal changes were measured in vivo using Doppler OCT technique (Doppler optical coherence tomography). The OCT system used in this study has axial resolution of 11 microm and lateral resolution about 14 microm in the cortical tissue. The velocity distributions along the vertical diameter of pial microvessels in a cranial window of the rats were measured at short time intervals by scanning the OCT sampling point repeatedly. The velocity profiles obtained in the pial arterioles were parabolic at any phase, although the centerline velocity pulsated following heart beats with amplitude as large as 50% of the temporal mean velocity. It indicates that the blood flow in the pial microvessels is a quasi-steady laminar flow, which is consistent with the flow expected for the case of a small Reynolds number and a small frequency parameter. The stimulus-induced increase in velocity pulsation was much larger than the increase in the mean velocity, which places a restriction on the mechanism of regulating the regional cerebral blood flow and blood volume. The results obtained in this study showed that the Doppler OCT has a potential of measuring velocity profiles and their temporal changes with both high temporal and spatial resolutions for the pial microvessels with diameter up to 200 microm.

Animals↗

PubMed2005

The neck domain of myosin II primarily regulates the actomyosin kinetics, not the stepsize.

In order to study the role of the neck domain of myosin in muscle contraction, we measured the steps of individual myosin II molecules engineered to have no neck domain (light chain-binding domain) by optical trapping nanometry. The actin filament and myosin cofilaments interacted on a glass surface to minimize the angle between them, and to minimize the interaction between myosin and the glass surface. The results showed that the average myosin stepsize did not change much when the neck domain was removed, but the sliding velocity decreased approximately fivefold. Furthermore, the duration of steps for neckless myosin was several times longer at saturated ATP concentration, indicating that the slower velocity was due to a slower dissociation rate of myosin heads from actin. From these data, we conclude that the neck domain of myosin-II primarily regulates the actomyosin kinetics, not the mechanics.

Actins↗

PubMed2005

Entropy rectifies the Brownian steps of kinesin.

Kinesin is a stepping motor that successively produces forward and backward 8-nm steps along microtubules. Under physiological conditions, the steps powering kinesin's motility are biased in one direction and drive various biological motile processes. The physical mechanism underlying the unidirectional bias of the kinesin steps is not fully understood. Here we explored the mechanical kinetics and thermodynamics of forward and backward kinesin steps by analyzing their temperature and load dependence. Results show that the frequency asymmetry between forward and backward steps is produced by entropy. Furthermore, the magnitude of the entropic asymmetry is 6 k(B)T, more than three times greater than expected from a current model, in which a mechanical conformational change within the kinesin molecular structure directly biases the kinesin steps forward. We propose that the stepping direction of kinesin is preferably caused by an entropy asymmetry resulting from the compatibility between the kinesin and microtubule interaction based on their polar structures.

Animals↗

PubMed2005

Paramagnetic artifact and safety criteria for human brain mapping.

Biological effects of magnetic field and their safety criteria, especially effects of gradient magnetic field on the cerebral and pulmonary circulation during functional brain mapping are still unclear. Here we estimated that magnetically induced artifacts for the blood oxygenation level- and flow- based functional magnetic resonance imaging are less than 0.1%, and disturbance in the pulmonary circulation is less than 1.3% even if the field strength of magnetic resonance system is risen up to 10 tesla. These paramagnetic effects are considered to be small and harmless during human brain mapping.

Editorial↗

PubMed2005

Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response.

Quantitative relationships between inputs and outputs of signaling systems are fundamental information for the understanding of the mechanism of signal transduction. Here we report the correlation between the number of epidermal growth factor (EGF) bindings and the response probability of intracellular calcium elevation. Binding of EGF molecules and changes of intracellular calcium concentration were measured for identical HeLa human epithelial cells. It was found that 300 molecules of EGF were enough to induce calcium response in half of the cells. This number is quite small compared to the number of EGF receptors (EGFR) expressed on the cell surface (50,000). There was a sigmoidal correlation between the response probability and the number of EGF bindings, meaning an ultrasensitive reaction. Analysis of the cluster size distribution of EGF demonstrated that dimerization of EGFR contributes to this switch-like ultrasensitive response. Single-molecule analysis revealed that EGF bound faster to clusters of EGFR than to monomers. This property should be important for effective formation of signaling dimers of EGFR under very small numbers of EGF bindings and suggests that the expression of excess amounts of EGFR on the cell surface is required to prepare predimers of EGFR with a large association rate constant to EGF.

Animals↗

PubMed2005

Trafficking of a ligand-receptor complex on the growth cones as an essential step for the uptake of nerve growth factor at the distal end of the axon: a single-molecule analysis.

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nm Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied <5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 microm2 s(-1). The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, approximately 4 microm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Animals↗

PubMed2005

Transcranial ultrasonography system for visualizing skull and brain surface aided by fuzzy expert system.

A conventional ultrasonography system can noninvasively provide human tissue and blood flow velocity information with real-time processing. In general, since the human skull prevents the disclosure of brain anatomy, we usually placed the sensor at the anterior and superior attachment site of the upper ear (the posterior temporal window) in adults. Due to this limitation, the conventional system cannot obtain transcranial information from arbitrary places in the skull. This paper describes a transcranial sonography system that can visualize the shape of the skull and brain surface from any point to examine skull fracture and brain disease such as cerebral atrophy and epidural or subdural hematoma. In this system, we develop anatomical knowledge of the human head, and we employ fuzzy inference to determine the skull and brain surface. To evaluate our method, three models are applied: the phantom model, the animal model with soft tissue, and the animal model with brain tissue. In all models, the shapes of the skull and the brain tissue surface are successfully determined. Next, the method is applied to two adults. As a result, we have determined the skin surface, skull surface, skull bottom, and brain tissue surface for the subjects' foreheads. Consequently, our system can provide the skull and brain surface information using three-dimensional shapes.

Algorithms↗

PubMed2004

EGF signalling amplification induced by dynamic clustering of EGFR.

Lateral interaction is an important feature of various types of cell surface receptors including the receptor tyrosine kinases (RTKs). Here we report that dynamic lateral interaction produces amplification and variation in signalling of the EGF receptor, a member of RTKs. Binding of EGF is known to induce transphosphorylation inside EGFR dimers. Using single-molecule techniques, the relationship between EGF binding and EGFR phosphorylation has been determined. The number of phosphorylated EGFR molecules became larger than that of EGF binding as unliganded EGFR was phosphorylated, meaning an amplification of EGF signalling. EGFR formed clusters continuously exchanging their elements through thermal diffusion, and direct and/or indirect lateral interactions. As a result, various types of activation sites differing in number of activated receptors were generated. Amplification required no cytoplasmic factors and was observed on semi-intact cells for a wide range of number of EGFR molecules (10(4)-10(6) per cell) suggesting generality of this process.

Adenosine Triphosphate↗

PubMed2004

The sensorimotor transformation of cross-modal spatial information in the anterior intraparietal sulcus as revealed by functional MRI.

The parietal cortex in monkeys and humans has been shown to play an important role in the transformation of sensory information to motor commands. However, it is still unclear whether in humans, these areas are divided functionally into subregions based on different combinations of sensory and motor modalities. To identify subregions in the parietal cortex involved in the sensorimotor information transformation between different modalities, functional MRI was used to examine brain areas activated during tasks requiring different sensorimotor transformations--i.e., various combinations of eye (saccade) or finger movements triggered by visual or somatosensory cues. We then compared the activations between cross-modal conditions (eye movements triggered by somatosensory cues and finger movements triggered by visual cues) and intramodal (eye movements triggered by visual cues and finger movements triggered by somatosensory cues) conditions. Although the parietal cortex was involved in all tasks regardless of sensorimotor combinations, the only region activated to a greater degree in the cross-modal conditions compared to the intramodal conditions was the anterior portion of the intraparietal sulcus (a-IPS). The results suggest that the a-IPS plays an important role in the sensorimotor transformation of cross-modal spatial information.

Adult↗

PubMed2004

A one-headed class V myosin molecule develops multiple large (approximately 32-nm) steps successively.

Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large ( approximately 36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP- and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (approximately 32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.

Actin Cytoskeleton↗

PubMed2004

Circulatory basis of fMRI signals: relationship between changes in the hemodynamic parameters and BOLD signal intensity.

Blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) is widely used as a tool for functional brain mapping. During brain activation, increases in the regional blood flow lead to an increase in blood oxygenation and a decrease in paramagnetic deoxygenated hemoglobin (deoxy-Hb), causing an increase in the MR signal intensity at the site of brain activation. However, not a few studies using fMRI have failed to detect activation of areas that ought to have been activated. We assigned BOLD-positive (an increase in the signal intensity), BOLD-negative (a decrease in the signal intensity), and BOLD-silent (no change) brain activation to respective circulatory conditions through a description of fMRI signals as a function of the concentration of oxygenated Hb (oxy-Hb) and deoxy-Hb obtained with near-infrared optical imaging (NIOI). Using this model, we explain the sensory motor paradox in terms of BOLD-positive, BOLD-negative, and BOLD-silent brain activation.

Adult↗

PubMed2004

Single-molecule imaging of the dynamic interactions between macromolecules.

In recent years, the development of single-molecule detection techniques has allowed the dynamic properties of biomolecules, which are normally obscured in conventional ensemble measurements, to be measured. One of these single-molecule detection techniques allows the measurement of dissociation and association events of individual molecules to be measured. This technique is based on the unique premise that the mobility between molecules that are bound and the mobility between those that are free in solution are different. The binding of ATP at the beginning and its dissociation at the end of the hydrolysis reaction were detected at the single-molecule level in real time. In this study, we extended this technique to image the dynamic interactions between large biomolecules (protein/protein and protein/polysaccharide). The binding and dissociation of fluorescently labeled macromolecules to partner molecules fixed on a glass surface were visualized by total internal reflection fluorescence microscopy. The dynamic interactions between the proteins in two energy conversion systems, that is, signaling proteins and enzyme molecules moving on dextran, have been measured. In these systems, the dynamic interactions were sensitive to the factors determining the chemical reactions. Thus, the dynamic interactions monitored in the single-molecule measurements provided useful information to further the understanding of the underlying mechanisms of energy conversion systems.

Adenosine Triphosphate↗

PubMed2004

Unconstrained steps of myosin VI appear longest among known molecular motors.

Myosin VI is a two-headed molecular motor that moves along an actin filament in the direction opposite to most other myosins. Previously, a single myosin VI molecule has been shown to proceed with steps that are large compared to its neck size: either it walks by somehow extending its neck or one head slides along actin for a long distance before the other head lands. To inquire into these and other possible mechanism of motility, we suspended an actin filament between two plastic beads, and let a single myosin VI molecule carrying a bead duplex move along the actin. This configuration, unlike previous studies, allows unconstrained rotation of myosin VI around the right-handed double helix of actin. Myosin VI moved almost straight or as a right-handed spiral with a pitch of several micrometers, indicating that the molecule walks with strides slightly longer than the actin helical repeat of 36 nm. The large steps without much rotation suggest kinesin-type walking with extended and flexible necks, but how to move forward with flexible necks, even under a backward load, is not clear. As an answer, we propose that a conformational change in the lifted head would facilitate landing on a forward, rather than backward, site. This mechanism may underlie stepping of all two-headed molecular motors including kinesin and myosin V.

Actins↗

PubMed2004

Absorber's effect projected directly above improves spatial resolution in near infrared backscattered imaging.

The spatial resolution of near infrared spectroscopic imaging in brain function mapping studies needs to be improved. Most near infrared spectroscopic imaging systems use optical fibers that are arranged like a lattice. The light source and the detector have one-on-one correspondence at intervals of about 3 cm. In this study, we apply several detectors against one source to improve spatial resolution. We assume that a local absorber within a strong scattering medium is a model for local cerebral activation. When we calculate absorbance, which is used to spectroscopically calculate hemoglobin concentration, the peak position shifts away from above the position where an absorber is located. As far as absorbance is calculated, we cannot obtain spatial information about the absorber even if many detectors are used against one source. When we calculate the difference between detected light intensities, however, we demonstrate that an absorber projected onto the measuring surface has an influence directly above it. We predict this property from a light diffusion equation and also prove it experimentally through measurements of a uniform resinous phantom with an absorber. This is one of the basic principles supporting the achievement of higher spatial resolutions with near infrared spectroscopic imaging.

Absorption↗

PubMed2004

Mechano-chemical coupling of molecular motors revealed by single molecule measurements.

Single molecule measurements have allowed series of kinetic events of biomolecules to be monitored without interruption. The stepwise movement of molecular motors was measured and analyzed in relation to the hydrolysis reaction of ATP. In the case of kinesin, forward and backward steps occurred stochastically at the same chemical state. The directional movement was explained by the asymmetric potential created by the interaction between kinesin and microtubules. Similarly thermal Brownian movement of myosin during the hydrolysis of single ATP molecules was biased through an asymmetric potential, resulting in directional movement. Thus, single molecule measurements have provided new approaches to analyze the function of molecular motors which often consist of several different events.

Actin Cytoskeleton↗

PubMed2004

In vivo imaging of the rat cerebral microvessels with optical coherence tomography.

A technique called optical coherence tomography (OCT) was applied to in vivo observation of microcirculation in the rat cerebral cortex. The OCT system used in this study provided cross-sectional images of the cerebral cortical tissue up to about 1 mm depth with longitudinal resolution up to 8 microm. It could visualize cross-sectional structure of the dura, arachnoid membrane, cortical tissue, and pial microvessels through the cranial window. Pial microvessels with diameter larger than several 10 microm could be detected to observe their cross-sectional shape, while the microvessels within the cortical tissue with smaller diameter were not discernible. The OCT observation revealed that the pial microvessels showed different spatial configurations depending on the cerebral preparations with intact dura and without dura. Stimulus responses of the somatosensory cortices were also different among the preparation methods; Delayed swelling of the cortical surface appeared in the somatosensory cortex following the electrical stimulation of the hind paw in the case of dura removal, which was restricted to a thin surface layer with less than several 10 microm. It is considered to reflect the reactive hyperemia accompanying the neuronal activation. Doppler frequency shift due to the blood flow was detected in pial arterioles. This phenomenon is promising to provide the velocity profile within microvessels and may be applicable to the functional imaging of the brain.

Animals↗