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Rat osteocalcin was subjected to proteolysis by cathepsins B and L at acid pH in vitro. Short fragments of fewer than 8 amino acids were liberated from both the NH2- and COOH-termini of the molecule, but the midportion, composed of antiparallel α-helical domains, was resistant to proteolysis. Intact rat osteocalcin bound 10 Ca2+/mol protein at pH 7.5 and the binding decreased to half that amount at pH 5.0, while the midportion fragment (Ala8-Lys43) bound 4–5 Ca2+/mol protein at both pH 5.0 and 7.5. When COOH-terminal-truncated rat osteocalcin (Tyr1-Lys43) was prepared with lysyl-endopeptidase, it showed nearly the same Ca2+ binding as that of the intact molecule. Our results suggest that proteolytic processing of the terminal sequence of osteocalcin alters its intrinsic Ca2+-binding capacity and that its NH2-terminal sequence is probably involved.

These effects of polychlorinated biphenyl (PCB) were examined by light and electron microscopy and biochemical analysis of lysosomal enzyme activities. Several experimental protocols with dosage schedules of either 0.2, 2.0, or 20 mg/kg of PCB were used. Typical histological changes were observed in mice given 2 mg/kg of PCB in a single injection. There were no remarkable changes until 4 days after PCB administration; marked cytoplasmic vacuolation was observed in parotid acinar cells at 7 days. The activities of lysosomal enzymes increased after the PCB injection and their maximum values appeared consistently at 4 days after the treatment; the increases were threefold for acid phosphatase, twofold for β-glucuronidase, threefold for cathepsin D, fivefold for cathepsin H and twofold for cathepsin L. As vacuolation was preceded by a large increase in lysosomal enzyme activities and the vacuoles co-localized with lysosomes, it is suggested that an increase in these activities induced by PCB may be closely related to the development of vacuolation in the parotid acinar cells as a subacute effect of PCB.

カテプシンDとEは, 主要な細胞内アスパラギン酸プロテアーゼである。本研究は, 両酵素の細胞内での活性調節機構を検索する目的で, 動物種や組織などの由来を異にする酵素を用いて, ATPや他のヌクレオチドによる酵素活性の上昇について比較, 検討を行った。ウシおよびラットカテプシンDは, ATPや他のヌクレオチド存在下では, ウシ血清アルブミンを基質としたときに顕著な酵素活性の上昇が認められ, AMP-PCP存在下でも酵素活性の上昇が認められた。ウシヘモグロビンや合成基質を用いたときは, アルブミンほどの酵素活性の上昇は認められなかった。一方, ヒト赤血球およびラット脾臓カテプシンEは, ヘモグロビンを基質としたときのみ, ATPや他のヌクレオチド存在下にて酵素活性の上昇が認められた。さらに, ATPは, カテプシンEの熱安定性を顕著に上昇させ, 酵素分子を安定化することが明らかとなった。

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

The effect of local administration of osteocalcin (OC) on experimental tooth movement was examined in the rat. The maxillary first molar was first moved mesially with an initial tipping force of 30 g with a closed-coil spring anchored to the incisor for 10 days (n = 48). Three experimental groups (n = 8) were injected with purified rat OC at doses of 0.1, 1, and 10 micrograms, respectively. The injection into the palatal bifurcation site of the first molar was repeated daily. The control groups (n = 8) were injected with rat serum albumin (10 micrograms), phosphate buffered saline (PBS), or were not injected. Tooth movement was evaluated daily by measuring the inter-cuspal distance between the first and the second molars on a precise plaster model. The cumulative tooth movement (mm) in the 1-microgram OC-injected groups was significantly more than that in all of the control groups on day 9. The rate of tooth movement (mm/day) showed periodical elevation, with high values on days 1, 4, 7, and 9. Acceleration of tooth movement by OC was significant in the early experimental period. Subsequently, acceleration of early tooth movement by OC was histologically evaluated (n = 40). Each of four animals from the control (PBS, n = 20) and the experimental (1 microgram OC, n = 20) groups was killed daily up to 5 days. A significantly larger number of osteoclasts accumulated on the mesial alveolar bone surface in the 1-microgram OC-injected group on day 3 than that observed in control group. These results suggest that administration of OC accelerates orthodontic tooth movement due to enhancement of osteoclastogenesis on the pressure side, primarily in the early experimental period.

The release of serotonin (5-hydroxytryptamine, 5-HT) in the dorsal hippocampus was measured using an in vivo microdialysis method in rats subjected to the Vogel type conflict test. The conflict situation significantly increased 5-HT release in the dorsal hippocampus. Midazolam (0.75 and 1.5 mg/kg i.p.) suppressed the dosage-dependently this increased 5-HT release, an inhibition closely associated with the attenuation of conflict behavior. These findings suggest that the activation of serotonergic neuronal activity in the dorsal hippocampus is linked to mediation of anxiety-related behavior.

The relationship between the morphological changes and vitamin A content during the development of acute toxicity induced by polychlorinated biphenyl (PCB) in mouse parotid glands was investigated. PCB was administered intraperitoneally at a single dose of 2 mg/kg. Ultrastructural studies revealed remarkable morphological changes in the rough endoplasmic reticulum, nucleus, Golgi apparatus and the secretory granules at 7 days after the administration of PCB. The activities of adenosine monophosphatase (AMPase) and alkaline phosphatase were increased 1 day after PCB administration. Then the activity of NADPH-cytochrome c reductase increased 4 days after PCB administration. Subsequently, the vitamin A content of the parotid glands significantly decreased at 7 days compared with the control. These sequential changes in enzyme activities implied that the decrease of vitamin A content in the parotid glands may be partly due to catabolism of vitamin A by increased activities of microsomal enzymes induced by PCB. In conclusion, it is suggested that PCB also induces drug metabolizing enzymes in the parotid gland cells and that the acute toxicity of PCB on this tissue may occur, at least partly, through the reduction of vitamin A not only by the secondary effect from liver impairment but also by the locally accelerated catabolism of vitamin A in the mouse parotid gland.

We investigated the effect of microtubule inhibitors on the amelogenesis in rat incisor enamel at the maturation stage by the glyoxal bis (2-hydroxyanil) (GBHA) staining method. Several red stripes stained with GBHA appeared on the maturation enamel surface of control rats. Colchicine injection (1.3 mg/kg, s.c.) disarranged the GBHA staining stripes and increased the staining area. The ratio of the GBHA staining area to the maturation enamel surface was about 25% in the control, but this value increased about 60% at 8 and 24 hr after the colchicine injection. Lumicolchicine, which does not have the ability to disrupt microtubules, did not cause any significant changes in the enamel surface. The incorporation of 45Ca to the maturation enamel was also reduced by colchicine. Since the hypocalcemic action of microtubule inhibitors may be related to the change of the enamel surface, other drugs, sodium salicylate and sodium fluoride, that have a hypocalcemic action were tested. The staining pattern was not altered by these drugs. Therefore, the hypocalcemic action was independent of the changes of the GBHA staining pattern. These results indicated that the disruption of microtubules in the ameloblasts inhibited calcium movement in the maturation enamel, resulting in the disarrangement of the GBHA staining pattern.

The enamel of the continuous growing incisors of adult rats is characterized by the presence of a orange-yellow pigmentation, which is caused by the content of large amounts of iron. Hi stochemical studies and electron microprobe analysis have shown that iron-containing pigment is also identified within the rat incisor ameloblasts during the enamel maturation stage [1–4]. Autoradiographic studies using 55Fe have demonstrated that the iron is incorporated from papillary layer to the ameloblasts and accumulated during the progress of maturation stage, and finally secreted onto the completely mineralized enamel surface at the end of maturation[5–7]. It is thought that iron is transported as the form of transferrin in the blood plasma and stored as the form of either ferritin or hemosiderin within the cells.

The quantitative effect of vinblastine, colchicine and lumicolchicine on the rate of dentine formation was determined by chronologically-labelling dentine with the lead salt of EDTA, before and after the intravenous administration of the test drug. Colchicine and vinblastine produced a dose-dependent inhibition of dentine formation. Lumicolchicine had no adverse effect. Although the log dose-response lines of colchicine and vinblastine were parallel, the effect of vinblastine was more potent. The relative potency ratio of vinblastine to colchicine was 1.25–1.70. These results suggest that both colchicine and vinblastine may act at the same site, i.e. microtubules, thereby inhibiting the secretion of new dentine matrix.

The action of microtubule inhibitors on dentine matrix formation, was investigated in the present study. The following experiments were carried out:(Exp.1) The effect of vinblastine (VB), colchicine(Col) and lumicolchicine(L-col) on the rate of dentine formation was determined by chronologically-labelling dentine with the lead salt of EDTA before and after the intravenous administration of the test drugs. (Exp.2) The effect of Col and VB on the incorporation of [3H ]-proline into odontoblasts and on the secretion of the radioactive materials from odontoblasts into dentinal matrices were studied autoradio-graphically at 2,4,8 and 24 hr after the injection of the test drugs. Col and VB produced a dose-dependent inhibition of dentine formation. No such effect was found in L-col, an isomer of Col lacking the ability to bind to microtubules. Although the log dose-response lines of Col and VB were parallel, the effect of VB was more potent. The relative potency ratio of VB to Col was estimated to be 1.25-1.70(95 percent confidence interval). These results suggest that both Col and VB may act at the same site, i.e. microtubules, thereby inhibiting the secretion of new dentine matrix. The results of Exp. 2, however, imply that there are some differences between Col and VB in the effects on the synthesis and the secretion of dentine matrix. The difference between the effect of the two drugs may depend on their different binding action with microtubules.