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Biomedical subjects

Y Takumi

Publications and source records attributed to Y Takumi.

At least 19 recordsLinked to original sources
PubMed2006

CRYM mutations cause deafness through thyroid hormone binding properties in the fibrocytes of the cochlea.

BACKGROUND: In a search for mutations of mu-crystallin (CRYM), a taxion specific crystalline which is also known as an NADP regulated thyroid hormone binding protein, two mutations were found at the C-terminus in patients with non-syndromic deafness. OBJECTIVE: To investigate the mechanism of hearing loss caused by CRYM mutations METHODS: T3 binding activity of mutant mu-crystallin was compared with that of wild-type mu-crystallin, because mu-crystallin is known to be identical to T3 binding protein. To explore the sites within the cochlea where mu-crystallin is functioning, its localisation in the mouse cochlea was investigated immunocytochemically using a specific antibody. RESULTS: One mutant was shown to have no binding capacity for T3, indicating that CRYM mutations cause auditory dysfunction through thyroid hormone binding properties. Immunocytochemical results indicated that mu-crystallin was distributed within type II fibrocytes of the lateral wall, which are known to contain Na,K-ATPase. CONCLUSIONS: CRYM mutations may cause auditory dysfunction through thyroid hormone binding effects on the fibrocytes of the cochlea. mu-Crystallin may be involved in the potassium ion recycling system together with Na,K-ATPase. Future animal experiments will be necessary to confirm a causal relation between Na,K-ATPase, T3, and deafness.

Animals↗
PubMed2005

Type IX collagen is crucial for normal hearing.

cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.

Animals↗
PubMed2001

Various glutathione S-transferase isoforms in the rat cochlea.

The localization of three glutathione S-transferase (GST) isoforms in the rat cochlea was examined using specific antibodies against each isoform. GST immunoreactivities were found in particular parts of the cochlea, including the intermediate cells and the basal cells of the stria vascularis and various types of fibrocytes in the spiral ligament. The different cell types showed varying combinations of GST isoforms. The GST immunopositive cells identified in the present study may play a central role in the metabolism and inactivation of endogenous and exogenous ototoxic compounds. The specific arrangements also indicated a possible contribution to the detoxification process in the form of a blood-labyrinth barrier.

Animals↗
PubMed2001

Connexin 26 distribution in gap junctions between melanocytes in the human vestibular dark cell area.

We have previously demonstrated the presence of gap junctions between melanocytes in the human vestibular organ and have speculated that melanocytes function in maintaining the homeostasis of the microenvironment of the inner ear. The purpose of the present study was to characterize the expression and ultrastructural localization of connexin (Cx) protein in melanocytes of the human vestibular organs. Surgical material was obtained from patients operated on for vestibular schwannoma and was processed for light microscopy, confocal laser scanning microscopy, conventional TEM, and immuno TEM. The specimens were labeled with anti-Cx26, Cx32, and Cx43 antibodies and examined by light microscopy. Specimens were also labeled with anti-Cx26 antibody and examined by laser microscopy and immuno-TEM methods. The specimens examined in this study were mainly dark cell areas from the human vestibular organ, whose epithelial and subepithelial layers are rich in melanocytes. Light-microscopic immunohistochemical studies showed positive labeling for Cx26 protein between subepithelial melanocytes, and Cx32 was also detected. Use of anti-Cx26 antibody and confocal laser scanning microscopy revealed high levels of Cx26 around the subepithelial melanocytes. Post-embedding immuno-gold transmission electron microscopy showed significant aggregation of gold particles (33.97 +/- 8.01% of total gold particles) around the gap junctions of the subepithelial melanocytes. The results of this study indicated that melanocytes are connected through gap junctions that mainly contain Cx26. This suggested that the melanocytes in the human vestibular organ may play a role in transporting material between the endolymph and perilymph.

Adult↗
PubMed2001

Neurotransmission in the vestibular endorgans--glutamatergic transmission in the afferent synapses of hair cells.

In the sensory pathways the first synapse is that between hair cells and primary afferent neurons and its most likely neurotransmitter candidate has long been thought to be glutamate. A number of pharmacological and electrophysiological studies have lent credence to this theory (reviewed by Bledsoe et al. 1988, Bobbin 1979, Ehrenberger and Felix 1991, Puel et al. 1991; Puel 1995) as has recent neurochemical and immunocytochemical work (reviewed by Ottersen et al. 1998; Usami et al. 2000). These recent studies reveal that the afferent hair cell synapse resembles the central glutamate synapses in many ways. Of the proteins confirmed to be involved in signal transduction and transmitter metabolism at most central synapses, many are also seen in the afferent hair cell synapse, and have an analogous compartmentation. On the other hand, there are also important differences, especially those related to the molecular mechanisms that underlie transmitter release.

Animals↗
PubMed2000

Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase.

Two pools of phosphate-activated glutaminase (PAG) were separated from pig and rat renal mitochondria. The partition of enzyme activity corresponded with that of the immunoreactivity and also with the postembedding immunogold labeling of PAG, which was associated partly with the inner membrane and partly with the matrix. The outer membrane was not labeled. PAG in intact mitochondria showed enzymatic characteristics that were similar to that of the membrane fraction and also mimicked that of the polymerized form of purified pig renal PAG. PAG in the soluble fraction showed properties similar to that of the monomeric form of purified enzyme. It is indicated that the pool of PAG localized inside the inner mitochondrial membrane is dormant due to the presence of high concentrations of the inhibitor glutamate. Thus the enzymatically active PAG is assumed to be localized on the outer face of the inner mitochondrial membrane. The activity of this pool of PAG appears to be regulated by compounds in the cytosol, of which glutamate may be most important.

Animals↗
PubMed1999

The arrangement of glutamate receptors in excitatory synapses.

Electron microscopic immunogold analyses have revealed a highly differentiated arrangement of glutamate receptors at excitatory synapses in the central nervous system. Studies focused on the hippocampus and cerebellum have shown that the postsynaptic specialization is the preferential site of NMDA and AMPA receptor expression, and that the delta 2 receptor is similarly concentrated at this site. In cases of colocalization (AMPA and NMDA, or AMPA and delta 2) the two receptor types appear to be intermingled rather than segregated to separate parts of the membrane. The different groups of metabotropic receptor exhibit distinct distributions at the synapse: group I receptors occur in membrane domains lateral to the postsynaptic specialization; group II receptors are expressed in preterminal membranes or extra-synaptically; whereas group III receptors are found in, or close to, the presynaptic active zone consistent with their roles as autoreceptors. The differentiated distribution of glutamate receptors reflects their functional heterogeneity and explains why some receptors are activated only at high firing frequencies.

Animals↗
PubMed1999

Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs.

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1-4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.

Animals↗
PubMed1999

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum.

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high-resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (approximately 96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved, the synthesis of transmitter glutamate may be less uniform than previously expected.

Animals↗
PubMed1999

Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study.

Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.

Animals↗
PubMed1999

Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.

Postembedding immunogold labeling was used to determine the relationship between AMPA and NMDA receptor density and size of Schaffer collateral-commissural (SCC) synapses of the adult rat. All SCC synapses expressed NMDA receptors. AMPA and NMDA receptors were colocalized in at least 75% of SCC synapses; the ratio of AMPA to NMDA receptors was a linear function of postsynaptic density (PSD) diameter, with AMPA receptor number dropping to zero at a PSD diameter of approximately 180 nm. These findings indicate that 'silent' SCC synapses are smaller than the majority of SCC synapses at which AMPA and NMDA receptors are colocalized. Thus synapse size may determine important properties of SCC synapses.

Animals↗
PubMed1998

Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.

The synapses between sensory cells in the inner ear and the afferent dendrites of ganglion cells are well suited to investigations of fundamental mechanisms of fast synaptic signalling. The presynaptic elements can be isolated for electrophysiological and functional studies while the synapses can be easily recognized in the electron microscope due to their distinct morphological features. This allows for a broader range of correlative functional and structural analyses than can be applied to synapses in the central nervous system (CNS). As in most fast excitatory synapses in the CNS the transmitter in the afferent hair cell synapses appears to be glutamate or a closely related compound. Recent studies have revealed many of the key molecular players at this type of synapse and how they are spatially and functionally coupled. By use of high resolution immunogold cytochemistry it has been shown that AMPA glutamate receptors are specifically expressed in the postsynaptic specialization of afferent hair cell synapses (except at those established by outer hair cells in the organ of Corti) and that their density varies as a function of the distance from the release sites (demonstrated for the afferent contacts of inner hair cells). The glutamate transporter GLAST is localized in supporting cell membranes and concentrated in those membrane domains that face the synaptic regions. Glutamine synthetase and phosphate-activated glutaminase--which are responsible for the interconversion of glutamate and glutamine--are selectively localized in non-neuronal and neuronal elements, respectively. Taken together with quantitative immunogold data on the cellular compartmentation of glutamate and glutamine the above findings suggest that the sensory epithelia in the inner ear sustain a cycling of glutamate carbon skeletons. In this process, the supporting cells may carry out functions analogous to those of glial cells in the CNS. Functional and morphological analyses of the presynaptic membrane indicate that L-type Ca(2+)-channels and Ca(2+)-activated K(+)-channels are colocalized and clustered at the active zone. Influx through the L-type channels triggers synaptic release and their close spatial association with Ca(2+)-activated K(+)-channels appears to be critical for frequency tuning. The focal expression of different Ca(2+)-channels combined with a high intracellular buffering capacity permits several Ca(2+)-signalling pathways to operate in parallel without undue interference. The molecular organization of the afferent hair cell synapses reflects the functional demand for speed and precision and attests to the ability of the pre- and postsynaptic elements to target and anchor key proteins at specific membrane domains.

Animals↗
PubMed1998

Select types of supporting cell in the inner ear express aquaporin-4 water channel protein.

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non-epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4-mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.

Animals↗
PubMed1998

Quantitative immunogold cytochemistry reveals sources of glutamate release in inner ear ischemia.

Glutamate is thought to be a major neurotransmitter between hair cells and afferent dendrites in the inner ear. However, excessive glutamate is known to be excitotoxic, and may be involved in ischemic neuronal damage in the central nervous system. The glutamate concentration in the perilymph has been reported to increase during ischemia, but the source of glutamate is still unclear. In the present study, we have used post-embedding immunogold cytochemistry to analyse changes in the cellular distribution of glutamate in the guinea pig organ of Corti during ischemia. The areal gold particle densities in the inner hair cells of the ischemic side were lower than those of the control side, indicating that glutamate may be released from the hair cells during ischemia. Adjacent supporting cells (border cells) also showed a decrease in particle density, suggesting that they constitute an additional source of glutamate.

Animals↗
PubMed1997

Cell death in the inner ear associated with aging is apoptosis?

Programmed cell death (apoptosis) in the inner ear of senescence-accelerated mouse was identified using specific labeling of fragmented DNA (the TUNEL method). In spite of some inter-individual differences, the apoptotic cells were predominantly found in the phylogenetically newer part of the inner ear, the cochlea and the saccules. In the saccules, sensory hair cells as well as supporting cells were positively labeled. In the cochlea, positive staining was detected in inner and outer hair cells, pillar cells, Deiters' cells, interdental cells, the stria vascularis (marginal cells, intermediate cells, basal cells), and cells in Reissner's membrane. The present results suggest that age-related cell death, which may cause hearing impairment and dysequilibrium, is due to apoptosis occurring in the inner ear.

Aging↗
PubMed1997

Discrete cellular and subcellular localization of glutamine synthetase and the glutamate transporter GLAST in the rat vestibular end organ.

Glial cells play an important role in the removal and metabolism of synaptically released glutamate in the central nervous system (CNS). It is not clear how glutamate is handled at peripheral glutamate synapses, which are not associated with glia. Glutamate is a likely transmitter in the synapse between the hair cells and afferent dendrites of the vestibular end organ. Immunocytochemistry was performed to investigate the distribution at this site of the high affinity glutamate transporter GLAST and glutamate metabolizing enzyme glutamine synthetase. Confocal microscopy revealed that GLAST and glutamine synthetase were co-localized in supporting cells apposed to the immunonegative hair cells. Postembedding immunoelectron microscopy revealed that GLAST was heterogeneously distributed along the plasma membranes of the supporting cells, with higher concentrations basally (at the level of the afferent synapses) than apically. Both immunoreactivities were also present in non-neuronal cells in the vestibular ganglion. The present findings suggest that glutamate released at the afferent synapse of vestibular hair cells may be taken up by adjacent supporting cells and converted into glutamine. Thus, at this peripheral synapse, the supporting cells may carry out functions similar to those of glial cells in the CNS.

ATP-Binding Cassette Transporters↗
PubMed1996

Effect of 4h preexercise high carbohydrate and high fat meal ingestion on endurance performance and metabolism.

The effect of a single high carbohydrate meal (HCM) and high fat meal (HFM) given 4 h before cycling exercises was determined for performance and metabolism. On the morning of the day of the trial, 10 endurance-trained collegiate males consumed either a single HCM (4669 kj; 79% CHO, 10% F and 11% P) or HFM (4711 kj; 30% CHO, 61% F and 9% P) as a 4 h preexercise meal. The intensity of exercise required was 65% of the maximal oxygen consumption (VO2max) for the first 120 min of exercise, followed by an increased dose of 80% VO2max. Endurance capacity did not differ between the HCM (128 +/- 3 min [SEM]) and HFM (122 +/- 3 min) trials. There was no significant difference in blood glucose levels in the HCM and HFM trials. A significantly higher (p < 0.01) serum insulin level in the HCM trials over the HFM trials was only observed at the start of exercise. Blood lactate levels (< 2.6 mM) during cycling remained below the anaerobic threshold. Respiratory exchange ratio in the HCM trials was significantly higher (p < 0.05 or p < 0.01) than that in HFM trials during the first 40 min of exercise. This was accompanied by significantly lower (p < 0.05 or p < 0.01) levels in serum free fatty acid and beta-HOB for the HCM trial. There was no significant difference between both diet regimens as to heart rate, oxygen consumption and perceived exertion during exercise. These results suggest that a single HCM and HFM given 4 h before exercise influences fuel utilization in the initial stages of prolonged cycling, but these meals may have little effect on endurance capacity.

Adult↗