Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes.

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.


Differential involvement of matrix vesicles during the initial and appositional mineralization processes in bone, dentin, and cementum.

The distribution of matrix vesicles and its role in biological mineralization were examined in bone and dental hard tissues of the rat after daily administrations of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP), a potent inhibitor of mineralization, for 7 or 14 days. Newly formed, nonmineralized matrices of the HEBP-affected bone and mesodermal dental hard tissues other than circumpulpal dentin contained numerous mineral-filled matrix vesicles (MV), randomly distributed throughout the collagenous matrix. The distribution density of the mineral-filled MV in the HEBP-affected matrices of calvaria, metaphyseal trabecular bone, alveolar bone, and cellular cementum ranged from 60 to 70 per 100 microm(2), and no statistically significant differences were noted among the values. In the HEBP-affected dentin, however, MV were located only in the nonmineralized matrix of mantle dentin and totally absent in the circumpulpal dentin layers. Instead, the HEBP-affected circumpulpal dentin contained a dense meshwork of noncollagenous matrix enriched with calcium and phosphorus. Comparable meshwork structures were undetectable in nonmineralized matrices of the other hard tissues affected by HEBP. These observations suggest that a certain population of MV (60-70 per 100 microm(2)) is involved in the process of appositional mineralization in most of the mesodermal hard tissues, in addition to their well-known role in initial mineral induction in these tissues. Circumpulpal dentin appears to be an exception, where MV are not required for the appositional mineralization process. Exclusive localization of dentin phosphoproteins in circumpulpal dentin layers must take place to facilitate appositional mineralization at the calcification front, in the absence of MV.


Demonstration of putative Ca-binding domains in dentin matrix of rat incisors after daily injections of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP).

In order to clarify the initial process of dentin mineralization, the inhibitory effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on dentin mineralization was investigated. Rats (100 g) were subcutaneously injected with HEBP (8 mg P/kg) for 7 or 14 d, and the incisors were processed for Ca histochemistry and/or electron microscopy. HEBP-treated incisors demonstrated ladder-like alternate rows of mineralized and non-mineralized dentin at the apical end. GBHA revealed moderate Ca reactions in the non-mineralized circumpulpal dentin matrix where electron microscopy revealed rich distribution of fine mesh-like electron-dense material. Non-mineralized mantle dentin matrix was negative for Ca but contained numerous matrix vesicles (MVs) filled with crystalline and/or amorphous mineral deposits. Mineralization of circumpulpal dentin occurred independently of MV-rich mantle dentin layer in affected specimens. Our data provide histochemical evidence of possible Ca-binding property of the circumpulpal dentin matrix and its absence in the mantle dentin where MV-mediated mineralization occurs. In the mantle dentin, HEBP does not interfere with crystal growth in MVs but inhibits its outgrowth after membrane rupture. It is proposed that circumpulpal dentin matrix has a potential to mineralize independently of MV-mediated mineralization of mantle dentin, although MVs determine the initial site and timing of dentin mineralization.


Homeobox gene expression during bone formation induced by BMP.

Annals of the New York Academy of Sciences


Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry

グリコーゲンを認識するモノクローナル抗体の作製と免疫組織学的観察.

The Journal of the Stomatological Society, Japan


An ultrastructural study of mitotic chondrocytes in the proliferative zone of the rat tibial growth plate.

Mitotic chondrocytes containing fairly prominent cell organelles were observed in the proliferative zone of the rat tibial growth plate by electron microscopy. During the later stage of mitosis, the endoplasmic reticulum was mostly vacuolated, and the Golgi stacks were replaced by spherical and cylindrical vacuoles and small vesicles. The cytoplasmic division began at early telophase. However, the cytoplasmic cleavage furrow was so narrow that dividing chondrocytes looked as if they were partitioned by a slit. This is probably one reason for the appearance of the binuclear cells that are occasionally observed in the tibial growth plate.


An ultrastructural study of the mitotic preosteoblasts in the primary spongiosa of the rat mandibular condyle.

In this study, we observed mitotic preosteoblasts that have the structural features of osteoblasts in the primary spongiosa of the rat mandibular condyle. The rough endoplasmic reticulum and the Golgi apparatus showed remarkable disorganization during mitosis. The Golgi saccules were replaced by groups of large vacuoles and small vesicles. The cisternae of the rough endoplasmic reticulum also were vacuolized. Since this disorganization occurred in conjunction with the formation of the mitotic spindle, it is probably related to the changes of the microtubular cytoskeleton. Further, secretory granules were arrayed along the mitotic spindle microtubules at the metaphase, and concentrated around the midbody at the telophase. These findings indicate a close relationship exists between secretory granules and microtubules.


Ultrastructural observation on matrix fibers in the condylar cartilage of the adult rat mandible.

In this study, ultrastructural and immunohistochemical studies were performed on the adult rat (15 weeks old) mandibular condyle, with particular attention to the matrix fibers in the condylar cartilage. The fibrous zone had thick collagen fibrils which formed fibril bundles. These collagen fibrils consisted mainly of Type I collagen. From the proliferative zone to the mature zone, the density of the collagen fibrils became higher. In the hypertrophic zone, thick collagen fibrils were formed around the chondrocytes. Immunohistochemical study indicated that these collagen fibrils consisted mainly of Type I collagen. Therefore, it was confirmed that the hypertrophic chondrocytes in this tissue had one of the osteoblastic phenotypes.

The Bulletin of Tokyo Medical and Dental University


Histological observation of large light cells that seem to be surviving hypertrophic chondrocytes in the rat mandibular condyle.

Large light cells (more than 20 microns dia), including some with mitotic figures, appeared to be surviving hypertrophic chondrocytes. Thus at least a few hypertrophic chondrocytes in the rat mandibular condyle may survive, be released into the primary spongiosa, and divide.


4. Expression of enamel protein hy the methods for in situ hybridiza tion and immunohistochemistry in the incisor of the amelogenesis imperfecta rat(ami)(8th Annual Meeting of the Society of Hard Tissue Biology July 24, 1999 in Asahikawa)