Biomedical subjects

S Komazaki

Publications and source records attributed to S Komazaki.

At least 37 records · Page 2Linked to original sources

PubMed1998

Structure and expression of mitsugumin29 gene.

Recently mitsugumin29 unique to the triad junction in skeletal muscle was identified as a novel member of the synaptophysin family; the members of this family have four transmembrane segments and are distributed on intracellular vesicles. In this study, we isolated and analyzed mouse mitsugumin29 cDNA and genomic DNA containing the gene. The mitsugumin29 gene mapped to the mouse chromosome 3 F3-H2 is closely related to the synaptophysin gene in exon-intron organization, which indicates their intimate relationship in molecular evolution. RNA blot hybridization and immunoblot analysis revealed that mitsugumin29 is expressed abundantly in skeletal muscle and at lower levels in the kidney. Immunofluorescence microscopy demonstrated that mitsugumin29 exists specifically in cytoplasmic regions of the proximal and distal tubule cells in the kidney. The results obtained may suggest that mitsugumin29 is involved in the formation of specialized endoplasmic reticulum systems in skeletal muscle and renal tubule cells.

Amino Acid Sequence↗

PubMed1998

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

Animals↗

PubMed1998

Mitsugumin29, a novel synaptophysin family member from the triad junction in skeletal muscle.

In skeletal muscle, excitation-contraction (E-C) coupling requires the conversion of the depolarization signal of the invaginated surface membrane, namely the transverse (T-) tubule, to Ca2+ release from the sarcoplasmic reticulum (SR). Signal transduction occurs at the junctional complex between the T-tubule and SR, designated as the triad junction, which contains two components essential for E-C coupling, namely the dihydropyridine receptor as the T-tubular voltage sensor and the ryanodine receptor as the SR Ca2+-release channel. However, functional expression of the two receptors seemed to constitute neither the signal-transduction system nor the junction between the surface and intracellular membranes in cultured cells, suggesting that some as-yet-unidentified molecules participate in both the machinery. In addition, the molecular basis of the formation of the triad junction is totally unknown. It is therefore important to examine the components localized to the triad junction. Here we report the identification using monoclonal antibody and primary structure by cDNA cloning of mitsugumin29, a novel transmembrane protein from the triad junction in skeletal muscle. This protein is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family. The subcellular distribution and protein structure suggest that mitsugumin29 is involved in communication between the T-tubular and junctional SR membranes.

Amino Acid Sequence↗

PubMed1998

Morphological abnormalities of adrenal gland and hypertrophy of liver in mutant mice lacking ryanodine receptors.

Ryanodine receptors (RyRs), which form Ca2+ channels in the membrane of the endoplasmic reticulum, consist of three subtypes (RyR1, RyR2, and RyR3). The RyRs release Ca2+ from the endoplasmic reticulum into the cytoplasm and thus play an important role, especially in the contraction of skeletal and cardiac muscle cells. The genes of these RyRs are also expressed in many non-muscle tissues, but the role played by RyRs in non-muscle cells is not fully understood. In the present study, we examined the morphological changes in such cells caused by a deficiency of RyRs genes using three mutant mice lacking RyR1, RyR3, or both RyR1 and RyR3. The results showed morphological abnormalities in the adrenal cortical cells in all three mutant mice. In addition, an excessive accumulation of glycogen granules in hepatic cells, and a hypertrophy of the liver were both present in those mutant mice lacking both RyR1 and RyR3. We discuss the relationship between the morphological abnormalities of the adrenal cortex and liver induced by a deficiency of RyRs, and the possible causes of these abnormalities.

Adrenal Cortex↗

PubMed1998

Effect of calcium on development of amiloride-blockable Na+ transport in axolotl in vitro.

The axolotl, Ambystoma mexicanum, which has no specific calcium-containing sieve layer in the dermis, provides useful material for the study of the effect of Ca2+ on the development of amiloride-blockable active Na+ transport across the skin of amphibians. We raised axolotls in thyroid hormone or aldosterone or cultured the skin with corticoid plus one of several Ca2+ concentrations and found that 1) although the short-circuit current (SCC) was increased by both aldosterone and 3,3',5-triiodo-L-thyronine in vivo, only corticoid was necessary for such an increase in vitro; 2) the development of the SCC in vitro was both corticoid and Ca2+ dependent, because the SCC was well developed with over 100 microM Ca2+ but not with under 10 microM Ca2+ in the presence of corticoid, nor even with 300 microM Ca2+ without corticoid; and 3) Ca2+, but not corticoid, was necessary for the formation of cell-to-cell junctions, because the resistance of the skin was well developed with 300 microM Ca2+ without corticoid.

Aldosterone↗

PubMed1997

Development of mechanisms regulating intracellular Ca2+ concentration in cardiac muscle cells of early chick embryos.

The development of mechanisms for the regulation of intracellular-free calcium ion concentration ([Ca2+]i) was investigated in precardiac mesodermal cells (PMC) and cardiac muscle cells (CMC) from early chick embryos by microfluorometry using a Ca2+-sensitive fluorescent probe, fura-2, and transmission electron microscopy. Microfluorometry indicated that two types of regulatory mechanisms, involving the dihydropyridine receptor (DHPR) and the ryanodine receptor (RYR), are present in CMC when the heartbeat begins at the 8-9 somite stages. Nifedipine completely suppressed the beating of hearts isolated from embryos on Days 1.5 and 2. Ryanodine had no effect on the beating of hearts isolated from embryos on Day 1.5, though it completely suppressed beating in hearts from Embryonic Day 2. Microfluorometry revealed that a change occurred in the Ca2+-regulating mechanisms of CMC on Day 2. Transmission electron microscopy showed the appearance in CMC, also on Day 2, of peripheral couplings with feet structures, and SR adjacent to the Z-line of myofibrils. These findings suggest that the calcium-induced calcium-release (CICR) mechanism appears in the CMC of the chick on the second day of embryonic development.

Animals↗

PubMed1997

Functional and morphological features of skeletal muscle from mutant mice lacking both type 1 and type 3 ryanodine receptors.

1. We generated mice with targeted disruptions in the genes for both ryanodine receptor type 1 (RyR-1) and type 3 (RyR-3) to study the functional roles of RyR subtypes in skeletal muscle. 2. In permeabilized myocytes lacking both the RyRs, the Ca(2+)-induced Ca2+ release (CICR) mechanism was completely lost, and caffeine failed to induce Ca2+ release. 3. Replacement of potassium methanesulphonate in an experimental intracellular solution with choline chloride resulted in Ca2+ release in the wild-type muscle but not in the mutant muscle lacking RyR-1. 4. The double-mutant mice exhibited more severe muscular degeneration than RyR-1-deficient mice with formation of large vacuoles and swollen mitochondria while structural coupling between T-tubules and the sarcoplasmic reticulum was retained. 5. These results demonstrate that CICR is mediated solely by RyR-1 and RyR-3 in skeletal muscle cells, and suggest that RyR-1 is involved in Cl(-)-induced Ca2+ release. The results also suggest the presence of molecular components other than RyRs responsible for the triad formation. RyR-3 may have a role in the normal morphogenesis of skeletal muscle cells, although functionally it can be replaced by RyR-1.

Animals↗

PubMed1997

Distribution and localization of galectin purified from Rana catesbeiana oocytes.

Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals. To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog. Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary. Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly. However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet. Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule. These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.

Amino Acid Sequence↗

PubMed1997

In vivo treatment of bullfrog tadpoles with aldosterone potentiates ACh-receptor channels, but not amiloride-blockable Na+ channels in the skin.

Amiloride-blockable Na(+) channels participate in active Na(+) transport across adult, but not larval, bullfrog skin. Their development is induced in vitro by culturing the tadpole skin with aldosterone. When tadpoles were raised in aldosterone (5 x 10(-7) M) for 2 weeks, however, neither development of such channels nor localization of antigen A, a marker of adult-type epidermis, was seen, the skin still being of the larval type. In contrast, aldosterone treatment did potentiate (by a factor of two) the activity of the acetylcholine receptor (ACh-receptor) channel, a functional marker of larval-type skin. The short-circuit current (SCC) across the skin, far from being inhibited by amiloride, was stimulated by both amiloride and ACh. The nystatin-stimulated SCC was about twice its control amplitude, suggesting that the aldosterone treatment also potentiated the activity of the Na(+) pump.

Aldosterone↗

PubMed1996

Occurrence of immunoreactive activin/inhibin beta(B) in gonadotrophs, thyrotrophs, and somatotrophs of the Xenopus pituitary.

An antibody against the Xenopus activin/inhibin beta(B) subunit (94-107) was raised in a rabbit. Using this antibody, the distribution of activin/inhibin beta(B) immunoreactivity in the pituitary of adult X. laevis was studied. Beta(B) immunoreactivity was detected in gonadotrophs, thyrotrophs, and somatotrophs under light microscopy. Electron microscopy revealed that a beta(B)-immunoreactive substance exists in LH, TSH, and GH granules, in contrast to findings in the rat and goldfish. These results indicate that the expression of activin/inhibin beta(B) in pituitary cells is not consistent among vertebrate species.

Activins↗

PubMed1996

Prolactin enables normal development of ACh-stimulated current in cultured larval bullfrog skin.

The response to acetylcholine (ACh) can be used as a marker for larval-type bullfrog skin because apically applied ACh induces an increase in short-circuit current (SCC) in larval-type but not adult-type skin. EDTA-treated larval skin, which contains only basal cells and does not respond to ACh, was used as the starting material for our culture. ACh, carbamylcholine, and choline stimulated SCC in skin that had been cultured with aldosterone (5 x 10(-7) M) supplemented with prolactin (PRL; 2 micrograms/ml). Atropine and d-tubocurarine each inhibited the ACh-induced stimulation of SCC in skin so cultured. Eserine, an inhibitor of acetylcholinesterase, also inhibited the ACh response. Amiloride stimulated SCC itself, but it reduced the ACh response. All of these results are quite similar to those seen in intact larval skin, suggesting that a larval-skin had differentiated from the basal cells used as the starting point for our culture. This is the first physiological report that PRL induces differentiation in vitro into a true larval-type bullfrog skin.

Acetylcholine↗

PubMed1996

Prolactin antagonizes the corticoid-promoted development of adult-type epidermis in cultured larval bullfrog skin.

EDTA-treated larval bullfrog skin, in which apical and skein cells had been removed and only basal cells remained, was cultured in one of four media. These contained either aldosterone (Aldo) or a mixture of Aldo, hydrocortisone (HC) and corticosterone (C), each either supplemented with prolactin (PRL) or lacking PRL. Skin cultured with Aldo alone or with the corticoid mixture (Aldo + HC + C) developed an adult-type epidermis: (i) both types of skin reacted to human blood group antigen A, a marker for the adult-type epidermis of bullfrog skin; (ii) amiloride decreased the short-circuit current Isc in these skin preparations, but acetylcholine (ACh) had no effect on the Isc. It seemed to make little difference to the results whether the skin was cultured with Aldo or with the corticoid mixture. PRL antagonized the action of Aldo and induced the development of a larval-type epidermis in both skin preparations: (i) the skin preparations did not react to human blood group antigen A; (ii) acetylcholine and amiloride each stimulated Isc in these preparations. Since ACh and amiloride each stimulated the Isc in skin with apical cells, ACh/amiloride-stimulated channels may be located on these cells.

11-Hydroxycorticosteroids↗

PubMed1996

Activin treated urodele ectoderm: a model experimental system for cardiogenesis.

The tissue interactions which comprise the inductive phenomena associated with urodele heart morphogenesis are relatively well understood. In order to take full advantage of the experimental potential of this system formulation of an in vitro tissue culture system would be very helpful. Herein are described conditions for culturing Cynops pyrrhogaster early gastrula ectoderm tissue in the presence of the peptide growth factor activin. Two-week old explant cultures frequently displayed beating heart-like rudiments within. The beating frequency was measured and the extent to which cytodifferentiation mimicked normal heart differentiation assessed. Both measurements provided optimistic assessments which should encourage further exploitation of this model system.

Activins↗

PubMed1995

Rat HPC-1/syntaxin 1A and syntaxin 1B interrupt intracellular membrane transport and inhibit secretion of the extracellular matrix in embryonic cells of an amphibian.

HPC-1/syntaxin 1A and syntaxin 1B are proteins that have been implicated in the docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane in neural cells. Capped RNAs (cRNAs) for rat HPC-1 and syntaxin 1B were injected into embryonic cells of an amphibian, the Japanese newt. The effects of the proteins translated from the injected cRNAs on intracellular membrane transport and secretion of the extracellular matrix (ECM) were then investigated. Immunoblotting and immunoelectron microscopy showed that the HPC-1 synthesized in the embryonic cells was localized on the membranes of Golgi complexes and vacuoles and on the plasma membrane. Electron microscopy revealed the morphological deformation of Golgi complexes, an appearance of large number of vacuoles, and the disappearance of the ECM from the cell surface in the cRNA-injected embryos. The results showed that HPC-1 and syntaxin 1B interrupt the pathways of intracellular membrane transport and inhibit the secretion of ECM by amphibian embryonic cells. Similar mechanisms may be involved in regulation of the secretory process of synaptic vesicles in mammalian neural cells and in regulation of intracellular membrane transport and constitutive secretion of ECM in amphibian embryonic cells.

Animals↗

PubMed1995

Calcium-containing, smooth-surfaced endoplasmic reticulum and vacuoles in cells of the blastopore-forming region during gastrulation of the newt, Cynops pyrrhogaster.

Ultrastructural changes in cells of the blastopore-forming region (BFR) were examined during gastrulation of the newt, Cynops pyrrhogaster. Smooth-surfaced endoplasmic reticulum (sER), tubular in shape and with two types of vacuole, empty vacuoles and multilamellar vacuoles, appeared in cells of the BFR at the beginning of formation of the blastopore. The extent of the tubular sER increased during formation of the blastopore. With the deepening of the blastoporal groove, the tubular sER and multilamellar vacuoles disappeared in cells of the BFR. The empty vacuoles increased in number and appeared throughout the cytoplasm of the cells in the BFR and the marginal zone as formation of the archenteron progressed. Many large empty vacuoles were closely associated with deformed lipid droplets. Cytochemistry and X-ray microanalysis revealed the accumulation of calcium in the tubular sER and in some of the empty vacuoles. These results suggest important roles for the calcium-containing tubular sER in the regulation of the intracellular concentration of calcium during formation of the blastopore, as well as a role for empty vacuoles in the dissolution and consumption of lipid droplets during early gastrulation.

Animals↗

PubMed1994

Localization of activin and follistatin proteins in the Xenopus oocyte.

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos.

Activins↗

PubMed1994

Calcium-containing vacuolated mitochondria during early heart development in chick embryos as demonstrated by cytochemistry and X-ray microanalysis.

The ultrastructure of mitochondria in the developing heart was examined in chick embryos from 2 to 7 days after fertilization. Vacuolated mitochondria were observed in the heart muscle cells of embryos at all stages examined. The number of vacuolated mitochondria as a percentage of total mitochondria in muscle cells was high in embryos at 3 and 4 days and was much higher in the ventricular cells than in the atrial cells. Examination by cytochemistry and X-ray microanalysis revealed the accumulation of calcium in the vacuoles of mitochondria. These results suggest important roles for vacuolated mitochondria in the regulation of the intracellular concentration of calcium during the early development of the chick heart.

Animals↗

PubMed1993

Ultrastructural localization of calcium in the chick yolk sac membrane endodermal cells as revealed by cytochemistry and X-ray microanalysis.

The yolk sac membrane (YSM) of the chick embryo transports calcium from the yolk into the embryonic circulation during the first half of development, but the intracellular pathway of calcium transport is poorly understood. In the present study, the ultrastructural localization of calcium was investigated in cells of the YSM of 9-day chick embryos. X-ray microanalysis as well as cytochemical techniques performed on yolk sac membrane cells treated with potassium oxalate, potassium ferricyanide and potassium antimonate demonstrated accumulation of calcium in yolk granules, digested yolk products, electron-dense bodies (EDBs; 100-400 nm diameter) and electron-dense granules (EDGs; 30-50 nm diameter). When strontium ions were injected into the yolk, they were incorporated into the endodermal cells and sequestered specifically in EDGs. From these results, we propose that calcium enters the endodermal cells by endocytosis of calcium-containing yolk granules, as well as through calcium channels in the apical cell membrane. In the cytoplasm, digested yolk products, EDBs, and EDGs act as sites of sequestration and accumulation of calcium. Extrusion of intracellular calcium into the extracellular space and embryonic circulation is accomplished by exocytosis of calcium-containing material and via an ion pump in the basal cell membrane.

Animals↗