Ken-ichi Kiyomiya

Mechanism of toxic action of fluoride in dental fluorosis: whether trimeric G proteins participate in the disturbance of intracellular transport of secretory ameloblast exposed to fluoride

Abstract In enamel fluorosis model rats treated with sodium fluoride, secretory ameloblasts of incisor tooth germs exhibited disruption of intracellular trafficking. We examined whether heterotrimeric G proteins participated in the disruption of vesicular trafficking of the secretory ameloblast exposed to fluoride, using immunoblotting and pertussis toxin (IAP)-induced adenosyl diphosphate (ADP)-ribosylation for membrane fractions of the cell. Immunoblotting of crude membranes, post supernatants of the ameloblast, with anti-Gi3/o and anti-Gs antibodies showed that Gi3 or Go proteins existed in the secretory ameloblast, but Gs protein did not. Immunoblotting of the subcellular membrane fractions indicated that the Gi3 or Go proteins were located in the Golgi membrane, but were not in the rough endoplasmic reticulum (rER) membrane. Autoradiograph of IAP-induced ADP-ribosylation, however, showed the existence of IAP-sensitive G proteins both in rER and Golgi membranes. Fluoride treatment decreased the G proteins bound to both membranes. These findings indicate that different G proteins, both of which are IAP-sensitive, are present in the rER and Golgi apparatus, and suggest that these G proteins participate in the disturbance of intracellular transport of the secretory ameloblast exposed to fluoride.

Proteasome is a carrier to translocate doxorubicin from cytoplasm into nucleus

When an effective concentration of doxorubicin (DXR) was added into L1210 of a mouse leukemia cell line, DXR was rapidly distributed much more in the nuclei than in the other organelle within a few minutes. A [14C]DXR-binding fraction was obtained from the cytosol prepared from L1210 cells. The fraction was adsorbed to hydroxylapatite matrix and eluted from the matrix by 50-150 mM potassium phosphate buffer. The fraction showed high DXR-binding and Suc-Leu-Leu-Val-Tyr-MCA-degrading activity. The binding of [14C]DXR was inhibited by unlabeled DXR. Gel chromatography of the fraction with Sephacryl S-300 separated two fractions of high molecular weight (Peak I, approx. 750 kDa) and low molecular weight (Peak II). Peak I showed proteolytic activity. [14C]DXR-binding Peak I had much higher affinity to DNA-cellulose than [14C]DXR-binding Peak II. [14C]DXR-Peak I complex also was retained into the nuclei isolated from L1210 cells, temperature-dependently. These results suggest that a specific carrier to translocate DXR from cytoplasm into nucleus exists in L1210 cell and the carrier is proteasome.

Proprioceptive afferents survive in the masseter muscle of trkC knockout mice

Peripheral innervation patterns of proprioceptive afferents from dorsal root ganglia and the mesencephalic trigeminal nucleus were assessed in trkC-deficient mice using immunohistochemistry for protein gene product 9.5 and parvalbumin. In trkC knockout mice, spinal proprioceptive afferents were completely absent in the limb skeletal muscles, M. biceps femoris and M. gastrocnemius, as previously reported. In these same animals, however, proprioceptive afferents from mesencephalic trigeminal nucleus innervated masseter muscles and formed primary endings of muscle spindles. Three wild-type mice averaged 35.7 spindle profiles (range: 31-41), six heterozygotes averaged 32.3 spindles (range: 27-41), and four homozygotes averaged 32.8 spindles (range: 26-42). Parvalbumin and Nissl staining of the brain stem showed approximately 50% surviving mesencephalic trigeminal sensory neurons in trkC-deficient mice. TrkC-/- mice (n = 5) had 309.4 +/- 15.9 mesencephalic trigeminal sensory cells versus 616.5 +/- 26.3 the sensory cells in trkC+/+ mice (n = 4). These data indicate that while mesencephalic trigeminal sensory neurons are significantly reduced in number by trkC deletion, they are not completely absent. Furthermore, unlike their spinal counterparts, trigeminal proprioceptive afferents survive and give rise to stretch receptor complexes in masseter muscles of trkC knockout mice. This indicates that spinal and mesencephalic trigeminal proprioceptive afferents have different neurotrophin-supporting system during survival and differentiation. It is likely that one or more other neurotrophin receptors expressed in mesencephalic trigeminal proprioceptive neurons of trkC knockout mice compensate for the lack of normal neurotrophin-3 signaling through trkC.

Influence of fluoride on secretory pathway of the secretory ameloblast in rat incisor tooth germs exposed to sodium fluoride

Abstract Fluoride, which is an environmental toxicant, is a potent inducer of mottled enamel in humans and rats. To define the influence of fluoride on the secretory pathway in enamel fluorosis, mottled enamel was induced in the incisor tooth germs of rats by subcutaneous injections of sodium fluoride for 4 days, and then morphological and cytochemical changes of the secretory ameloblast were examined in the tooth germs with HRP-labeled lectin (Con A, GS-I, SBA and PNA) and En3 antibody labeling amelogenins. The accumulation of small vesicles on the route of the secretory pathway between the rER and the Golgi apparatus, disorder of Golgi stacks, and formation of abnormal large granules in distal cytoplasm were seen in the secretory ameloblast. Lectin staining patterns of the secretory ameloblast indicated the disturbance of the vesicular transport between the rER and the Golgi apparatus, and disorganization of the Golgi stack. Immunolabeling of the cell showed disruption of the sorting and fusion process on the secretory pathway. These results suggest that the fluoride disturbs the intracellular transport in the synthesis-secretory pathway of the ameloblast, and that this effect of fluoride on the synthesis-secretory pathway participates in the formation of enamel fluorosis.

Ruffini endings are absent from the periodontal ligament of trkB knockout mice

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.

Fluoride-induced ultrastructural changes in exocrine pancreas cells of rats: fluoride disrupts the export of zymogens from the rough endoplasmic reticulum (rER)

Abstract Influence of fluoride on exocrine pancreas cells was examined morphologically with traditional and prolonged osmium fixation techniques for electron microscopy in the enamel fluorosis model rats injected subcutaneously twice a day with 20 mg/kg body weight of sodium fluoride. Although the rough endoplasmic reticulum (rER) of exocrine pancreas cells in control rats was laminated and oriented parallel to the circumference of the nucleus, the rER of the cells in NaF-treated rats was dilated, disrupted the laminated arrangement, and changed to the globular-shape rER. Many intracisternal granules were formed in these globular-shape rER of the cells exposed to fluoride. Lots of autophagosomes were also seen in the exocrine cells with NaF treatment. The autophagosomes were limited with a double or multiple membranes, and contained cytoplasmic organelles and/or the intracisternal granules. The outer and inner leaflets of double membranes of the autophagosomes were usually separated by a distinct electron-lucent area. In prolonged osmium fixation, the area between the double membranes of the autophagosome was filled with osmiun reaction deposits. Many autophagosomes were encircled with the single or multiple osmiophilic layers. In some cases, the osmium positive saccules also surrounded the free surface of the globular-shape rER containing intracisternal granules. These findings indicate that fluoride disrupts the export of zymogens from the rER, resulting in formation of intracisternal granules and autophagosomes, and that the osmiophilic saccules participate in sequestration of cytoplasmic organelles in forming autophagosomes.

Mitochondrial cytochrome c oxidase as a target site for cephalosporin antibiotics in renal epithelial cells (LLC-PK1) and renal cortex

We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.

Effect of BDNF depletion on the formation of Ruffini endings in vibrissa follicles and the survival of their mechanoreceptive neurons in trigeminal ganglion.

We examined the influence of BDNF depletion in peripheral tissues on the formation of Ruffini endings and their neuronal survival by injections of neutralizable anti-BDNF antibody into mouse mystacial pads for periods of 5 days at different developmental stages of the Ruffini endings (the pre-formation stage from the 2nd to 6th day after birth, the formation stage from the 4th to 8th, or the post-formation stage from the 10th to 14th). The treatment at the pre-formation and formation stages caused a significant decrease in the number of Ruffini endings in vibrissa follicles. This decrease in Ruffini endings was accompanied with a significant increase in neuron apoptosis in the trigeminal ganglion (TG) in both stages. However, at the post-formation stage, the anti-BDNF injection showed no effect on the formation of the mechanoreceptors nor their neuronal survival. In the post-formation stage, the axoplasmic spins of Ruffini endings were circumferentially embraced with the cytoplasmic processes of terminal Schwann cells. The present study indicates that target-derived BDNF is essential for survival of mechanoreceptive nerves in the pre-formation and formation stages, but not in the post-formation stages of their development. It seems that Schwann cells participate in this switch-over of neuronal dependency on brain-derived neurotrophic factor.