Importance of non-collagenous proteins, especially osteocalcin and osteonectin in calcification was reviewed.
Osteocalcin is a γ-carboxyglutamic acid-containing protein in bone and synthesized by osteoblasts vitamin K-dependently. Osteocalcin has characteristic properties of Ca-binding and also adsorption to hydroxyapatite crystal. These properties and the appearance in developing bone strongly suggest some regulatory roles in bone calcification process.
Osteonectin is another bone-specific protein which has a property to bind selectively to both hydroxyapatite and type I collagen. This protein is found to be present in newly calcified bone tissue and in osteoblasts but not in soft tissues by immunofluorescent detection. These studies suggest that osteonectin may play a role in initiating calcification.

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A palaeopathological study concerning localized lesion with bone defect was made through gross examination of 17 jaws culled from 190 Japanese jaws (105 skulls) dating back from Jomon to Edo periods. Localized lesions with bone defects were found on 25 sites of 17 jaws, including 22 periodontal lesions (periapical abscess, apical granuloma and radicular cyst) of 16 jaws. Two cases of periapical lesions, which are thought to have been caused by severe attrition complicated by pulp cavity exposure, were seen in those of the Jomon period. Other interesting lesions diagnosed palaeopathologically as primordial cyst, Stafne's idiopathic bone cavity and subacute osteomyelitis with sequestrum were discribed, and the differential diagnoses concerning these lesions were discussed by use of macroscopic and roentogenologic findings.

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The purpose of this study is development of biological active compounds with tertiary carbon atom at 4-position of piperidine ring in 4-phenylpiperidine derivatives.
It has been previously reported that 4-phenyl-N-(3′-dialkylamino-2′-hydoxypropyl) piperidine derivatives (I) showed strong infiltration anesthetic and anti-inflammatory action.
In order to develop the active compounds in topical anesthetic action, amino alkanol derivatives (II) that substituted lipophilic methyl, phenyl and phenoxymethyl groups for 3′-dialkylamino methyl groups at (I), and amino alkanone derivatives (III) as oxidized form of (II) were synthesized.
The experimental results of local anesthetic action were as follows.
(1) IIa, b Ina, c showed strong infiltration of anesthetic action and excelled lidocaine·HCl in duration, degree of infiltration and appearance and disappearance of anesthetic action.
Furthermore, since no intradermal irritation, necrosis or redness were observed, Ina and IIIa, seemed to be highly interesting compounds as infiltration anesthetics.
(2) IIa, IIb, c showed stronger than (I) and cocaine·HCl, but showed slight intradermal irritation and necrosis.

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Epithelial cells from a neonatal rat tongue were successfully grown in cell culture. The isolated epithelial cell culture is described in association with the patterns of growth and maturation difference, formation of keratohyalin granules and keratinization. The density and size, distribution of the gap junctions and desmosome were computed planimetrically by the freeze-fracture method. The cell doubling required about 30 hours. The subculture of rat lingual epitheliums derived from primary explants was conducted upto 8 passages. Maturation and vertical stratification of the culture cells occurred at a later stage, demonstrated by an increase in cell organelle (rough endoplasmic reticulum, mitochondria and Golgi complex), tonofilament accumulation and presence of keratohyalin granules. An amorphous substance positive to ruthenium red stain and usually present in the space between the basal layer and plastic flask and hemi-desmosome structure were shown. No membrane coating granules were detectable. In freezefracture replicas, density and size of gap junction and desmosome increased with proliferation, differentiattion and maturation of the cells.

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The density and affinity of a-adrenergic receptors and their subunites were determined in the gland membrane preparations obtained from two to eight-week-old rats, using radioligands [³H]-dihydroe-rgocryptine ([³H]-DHE), [³H]-WB-4101 ([³H]-WB) and [³H]-clonidine ([³H]-Clonid) for the measurement of α-adrenergic, α1-adrenergic and α2-adrenergic receptors, respectively. During weaning, the rat were fed on solid chow (CE-2) and water ad libitum.
Scatchard analysis of saturation curves for adult membranes gave dissociation constants (KD) and the density of binding sites (Bmax) of 0.81nM and 77.3pM for [³H]-DHE, 0.29nM and 68.3pM for (³H)-WB and 0.52nM and 7.4pM per mg of membrane protein for [³H]-Clonid, respectively. In immature animals, the Bmax of [³H]-DHE and [³H]-Clonid were high in two-week olds, but decreased rapidly in three weeks. These values were reincreased significantly by four weeks and then declined thereafter. The bindings reached adult density at around eight weeks.[³H]-WB binding sites increased steadily during the developmental course. The KD for each ligand was low in two-week-old rats, but differed in the course of functional maturation in Bmax values during development.These findings suggest that each of the autonomic receptors in growing rat submandibular glands follows its own specific developmental process and that the appearance of an individual receptor subunite may be correlated with the ability of the developing gland to respond to cell proliferation, differentiation and transformation especially during the weaning period.

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In order to have the cell numbers counted with an autoelectric cell counter correspond to those with a microscope, discrimination of the autoelectric cell counter was modified by referring to the distribution of cell particles in a suspension medium. With this modification, the autoelectric cell counter was able to accurately count the cell numbers in the cell suspension medium.
The survival rate of the cells in the suspension medium was also studied. The survival rate of cells, which were separated from culture plate using emzymes such as pronase (0.05%), trypsin (0.25%), trypsin (0.05%) +EDTA-2Na (0.02%), collagenase (0.1%), dispase (500U/ml) was about 90-100%. These results suggested that about 0-10% of the cells counted with the autoelectric cell counter were dead cells and the these cells could lead to an erroneous cell count by the autoelectric cell counter.

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Diurnal changes in the labelling index (L. I.) and the mitotic index (M. I.) as well as the generation time of cells in the basal layer of the tongue epithelium of C₃Hf/He mice were observed by using the pulse labelling method with ³H-thymidine injection. Most of the labelling experiments were done at night when high labelling indices were available. The results were as follows: 1) The diuthal changes in L. I. and M. I. were clearly shown with the peak time at 17:00 for L. I. and at 3:00 for M. I. of cells in the basal layer. In the basal layer there was a local difference in the rat of L. I., namely, a high L. I. was obtained in close proximity to the posterior region of the papillae, and a low L. I. in the anterior region. The possibility of the existence of EPU (Potten; 1979) was suggested.
In general, it was assumed that the proliferation activity of basal cells was higher at night than in daytime. 2) The cellular generation time of basal cells was estimated by two different methods.The generation time obtained by PLM analysis, a method developed by Mendelsohn & Takahashi (1972), was estimated to be 33.6 hrs, including 6.1 hr for S, 3.6 hr for G₂+M and 23.9 hr for G₁. This was about 5 hr shorter than that obtained by daytime labelling experiments. However, it was found that the method of Brown & Oliver (1968) was not applicable for the present experimental results, because the NB/NT values did not break down at 0.5 as they did in the case of hamster cheek pouch cells. The reduction of NB/NT kept its linearity to a point less than 0.37. 3) From the results obtainedin the present study, the cellular event that is most closely related to diurnal changes in cell proliferation is the time whee cells leave the basal layer, which was about 8 hrs at night and 14.5 hrs in daytime after labelling. It seems reasonable to assume that most parts of S-cells in the basal layer do not enter the phase of differentiation (upper layer of the epithelium) immediately and directly after cell division. Some regulatory mechanisms may be intervening to control the transition of basal cells to the differentiation phase.

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Labelling indices (L. I.), cell generation time, and turnover time of basal cells in three different portions of the tongue epithelium were investigated by autoradiography technique in C3Hf/He mice.F or convenience, 3H-thymidine was injected in labelling the basal cells at night, 22: 00-23: 00, when a high rate of L. I. was obtained because of its diurnal changes. The cell generation time was determined by PLM analysis, developed by Mendelsohn & Takahashi. Brown & Oliver's method was also applied for determination of the cell turnover time.
The results suggested that there was some difference in the cell proliferatio n activity between threediff erent parts of the tongue anterior, middle, and posterior regions. In general, it can be stated that the posterior region retained higher activity in terms of cell proliferation than the anterior region. This can be supported from the results that cells in the posterior region showed a higher labelling index and a shorter cell generation time. Moreover, the turnover time of cells in the anterior region was seen to be longer than that of other regions. These results favour the view that there is some local difference in the activity of cell production in the tongue epithelium of C3Hf/He mice.

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To study the fact that Streptococcus mutans predominates in place of Strepto coccus sanguis in dental plaques on the teeth initiating dental caries, isolation of lytic bacteria against Streptococcus sanguis from dental plaque was examined. Lytic abilities of bacteria were demonstrated by clear lytic zones around the colonies on an agar plate containing heated streptococci cells. A number of lyt c bacteria against Streptococcus sanguis ATCC 10558 strain was isolated from infant dental plaque samples on the pit and fissure of second milk molar experienced caries of degree CI and of intact antagonistic molar.S treptococcus mutans was also predominantly isolated from the same plaque samples on pit and fissures, but not on other surfaces of the same molars and of deciduous central incisors, on which Streptococcus sanguis was detected as major streptococci.
All of lytic bacteria indicated the same characteristics and identified as Streptoc occus mutans on the basis of morphology of cells and colonies on Mitis-Salivarius agar plate, various biochemical tests, adherence abilities to smooth surfaces and productivity of insoluble polysaccharides. These lytic bacteria showed high lytic activities against only Streptococcus sanguis in the several species of streptococci isolated from the same oral cavity and against strains of cariogenic Streptococcus mutans regardless of serotypes, butl ow activities against themselves. Several strains of cariogenic Streptococcus mutans also showed lytic abilities against Str. sanguis strains. These findings suggest that lytic bacteria, havingthe characteristicsof Stretpcoccus mutans, may have taken part in the change ofthe species of streptococci in the human dental plaques at the time dental carieswas initiated.

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Lytic enzyme against the strains of Streptococcus sanguis ATCC10558 and Streptococcus mutans E-49 was obtained from the supernatant of culture of Streptococcus mutans AL7-1 strain isolated from infant oral cavity, forming clear lytic zones around the colonies on an agar plate containing heated cells of Streptococcus sanguis and identified as Streptococcus mutans by its various characteristics. The crude lytic enzyme showed optimal activity at pH 6.5 and at 42°C, pH and heat stability at range 5.2 to 7.0 and up to 45°C respectively. The lytic enzyme activity was accelerated by addition of Ca²⁺, Mn²⁺, Mg²⁺ and Pb²⁺ ions, but completely inhibited by addition of Cu²⁺ and Hg²⁺ ions. The lysozyme and caseinase activities of this lytic enzyme preperation were minor. The maximum yield of lytic enzyme activity was obtained from the 24h-culture of Streptococcus mutans AL7-1 strain grown in TM meium containing 0.1% cells (wet weight) of Streptococcus mutans E-49 strain and 0.2% sucrose, and having initial pH range 7.5 to 8.0.

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Aminopeptidase activity of Mycoplasma salivarium, measured by using L-leucine p-nitroanilide (LeuNA) as a substrate, was enhanced remarkably by 1 mM MnCl2 and slightly by 1 mM MgCl2, but not at all by 1 mM CaCl2. In addition, consistency of preincubation time of the crude enzymes (disrupted M. salivarium cells) with MnCl2 was demonstrated to be essential for assaying the activity quantitatively. The crude enzymes hydrolyzed LeuNA, ArgNA, LysNA and AlaNA, in a decreasing order, but not GlyNA and ProNA. Thus, LeuNA was shown to be the best of all tested substrates for activity assay.
M. orale ATCC 15539, M. buccale IID 802, M. faucium IID 996, M. hominis IID 801, M. fermentans IID 812, M. pneumoniae IID 815 and IID 817 were also found to possess aminopeptidase activity.

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Structural changes appearing in the upper alveolar bone and the neighboring bones due to traumatic occlusion were examined by use of the labelling method and microradiography. Ten adult dogs were kept for 29-141 days after respectively setting a high cast crown on the lower first molar. During the experimental period, the dogs were injected with tetracycline and calcein continuously, as shown in Fig. 1. After sacrificing, large frontal ground sections were made of the upper fourth premolar and first molar. The two images obtained by microradiography and fluorescence microscopy from the same ground sections were compared precisely.
Shortly after the high cast crown was set on antagonist, active remodeling became observable at the alveolar bone proper along the periodontal space of the upper alveolar bone given the excessive occlusal stress, and then, the arrangement of bone trabeculae became irregular and porous (Figs. 3-8). On the other hand, in the spongy bone, internal remodeling in which bone formation is much more active than resorption took place immediately after the experiment commenced and the bone trabeculae became thicker and its distribution denser (Figs. 3-8). However, these changes began to cease gradually after 7-8 weeks. At the compact bone adjacent to the orbita, stratified formation of bone began to appear at its orbital side, but this change ceased after 7-8 weeks (Figs. 3-8).
In some animals, active bone formation was observed in various parts of the zygomatic bone and perpendicular plate of the palatine bone, whereas no particular change was observed in the horizontal plate of the palatine bone (Fig. 4).

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The purpose of present study was to observe the changes appearing in the mandibular bone structure after many teeth were extracted. The dogs, all of whose lower premolars and molars on one side were extracted at the same time were kept for 57-181 days. The animals were injected continuously with tetracycline during the first half of the experimental period and with calcein during the latter half, as shown in Fig.1. Buccolingual ground sections of the mandibular bone (Fig.2) were first microradiographed and, then, subjected to fluorescence microscopy. The two images were compared precisely for the control and experimental sides.
The alveolar bone proper, spongy bone and newly formed bone in the socket became very porousseco ndarily within about 100 days. Resorption of the residual alveolar ridge progressed more extensively on the buccal than lingual side (Figs.4, 5, 7, 8-10, 12, 15, 17).
Changes in the bone formation became observable also on the entire outer surface of the compactb one, although they were different on the buccal and lingual sides and mesial and distal portions of the premolar-molar region. In the mesial portion, bone formation was accelerated on the buccal surface but retarded on the lingual surface. However, in the distal portion, active bone formation appeared on the lingual surface and resorption on the buccal surface (Figs.4-7, 10-18).
In the inner space surrounded by compact bone, two different changes were observed. One was the upward migration of the upper margin of the mandibular canal (Figs. 4, 5, 7-13, 15, 16, 19) and another was the active formation of bone on the entire surface of the inner wall of the com pact bone (Figs.4-7, 9 -16, 19). Internal remodeling in a form of replacement osteon was accelerated within the compact bone, especially its upper half (Figs.4, 6, 10, 11, 13, 14, 16). These changes observed in the compact bone on the experimental side seem to be related to distortion of the body of the mandibular bone occurring due to masticatory stress.

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Morphological observations were carried out on the lingual gland of Onychodactylus japonicus and the findings in the 60th stage with gills and the 70th stage without gills were compared. The fine structure of acinar cells of the lingual gland was approximately the same between the 60th and 70th stage. While homogeneous (Type A) secretory granules and those with core and hallo formation (Type B) werefound in the 60th stage, only Type A was found in the 70th stage.
Basal cells were frequently encountered in the 60th stage but only rarely in the 70th stage.
In myoepithelial cells, the development of intracellular myobundle was more pronounced in the 70th than in the 60th stage.

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The purpose of this investigation was to study the effect of sonicate supernatant from A. viscosus of ATCC 15987 strain on dissolution of rat alveolar bone powder by mouse peritoneal macrophages. The peritaneal cells (PEC) were induced by 10% proteose peptone. Macrophages in the PEC were isolated by using a method of rinsing the cultures in multiwell plates after one hour and again after 24 hrs. Then, sonicate supernatant of A. viscosus and rat alveolar bone powder in Ham's medium (supplemented with 5% fetal calf serum and HEPES) were added to the wells which cultured the macrophages. As a contrast, sonicate supernatant of A. viscosus alone or the bone powder alone was added to the wells. The number of adherent cells in the wells was calculated with an objective micrometer in a phase contrast microscope. Ca release from alveolar bone powder in the medium was measured by means of an inductively coupled plasma spectorometer. The cells cultured with bone powder and cell alone decreased remarkably in number with the passage of time. However, the decrease in the number of cells in the medium supplemented withsupernatant from A. viscosus was comparatively small. The addition of supernatant from A. viscosus into the culture medium promoted the dissolution of alveolar bone powder by macrophages.

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Although studies in recent years have shown that the bone tissue is dissolved bycultured macrophages, the conclusions are not definite enough to regard them as mechanisms of bone tissue dissolution. In this study, therefore, the effect of glucose concentration on the dissolution of bone tissue in medium cultured macrophages was examined by using a method in which the increase of Ca concentration in cultured medium which released Ca from the apatite in bone tissue by macrophages was measured. When glucose concentrations in a cultured medium were raised, the increase in concentration of lactate produced in a medium of p H 7.2 was accompanied by increased Ca release under experimental conditions with the same cellnumber. Consequently, the apatite solubility of macrophages was influenced by the degree of glucose concentration in a medium. It seems that increase of lactic acid produced from macrophages brings about Ca release from the apatite.

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A frog has numerous small teeth in the upper jaw, which form an upper dental arch. However, there is no periodontal ligament in a frog. Inside of the dental arch, the palatal mucosa forms a ridge which is called the palate shelf. In the present experiment, a new jaw reflex was found which was evoked from the palate shelf. Transient jaw closing reflex was elicited by mechanical stimulation of the rapidly adapting mechanoreceptors low threshold in the palate shelf. On the other hand, mechanical stimulation of the slowly adapting mechanoreceptors high threshold inhibited the activity of the jawclosing muscle.
Electrical stimulation of afferent nerve fibers of the palate shelf (palato-nasal nerve) also elicited reflex responses similar to those by mechanical stimulation. When weak electrical stimuli (0.4V) were applied to the palato-nasal nerve, a jaw closing reflex was observed during the stimulation. On the contrary, evoked activity of the jaw-closing muscle was inhibited by strong electrical stimuli (0.9V) applied to the same nerve. Excitatory reflex response was clear at a stimulus frequency above 10 Hz. Clear inhibitory reflex was observed at a stimulus frequency above 20 Hz.
The palate shelf is very sensitive to the touch and takes part in the reflex regulation of the jaw movement. In addition, the role of the palate shelf is functionally similar to that of the periodontal ligament. It was discussed that the jaw closing reflex in the present study was analogous to the periodontal-masseteric reflex in warm-blooded animals.

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In order to clarify the fiber architecture in the periodontal ligament of continuously-erupting tooth and to ascertain the existence of “intermediate plexus” reported by Sicher, the periodontal ligament of a rat incisor was examined by light and scanning electron microscopy.
The periodontal ligament of the rat incisor was composed of two different fiber elements, parallelorientedfiibers along the long axis of the tooth and transvers fibers running across the periodontal space from the cementum and alveolar bone.
The fine parallel-oriented fibers scattered in the periodontal space at the developmental site, gathered together gradually in the mid-region of the space near the mesial site of the second molar, increasing in size and number, and formed flat “sheet-like” bundles.
The transverse fibers arising from the cementum and alveolar bone respectively, extended into the mid-region of the periodontal space and formed flat “sheet-like” fiber bundles near the secondmolar similar to parallel-oriented fibers. Thereafter, both bundles were joined at the mesial site of the first molar.
Though parallel-oriented fibers were prominent near the base of the incisor, transverse fiber bundles replaced them at the crest of the alveolar process. These transverse bundles seem to play the role of support for the tooth in the bony socket.
An “intermediate plexus” reported by Sicher is a fictious image arising out of the plane of section.

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This experimental assay essentially measured the misincorporation of dGTP and dCTP into poly (dT) oligo (dA) template-primer, intact or UV-irradiated, to produce thimine dimers. The temperature inducible tif mutant cell-free extract was used for DNA polymerase in this assay. A “tif 42°C”conditions can be referred to as SOS⁺ and “tif 30°C” conditions as SOS-. Each reaction mixture (0.3ml) contained: 75mM Tris hydrochloride, pH 8.6 (0.2mM MnCl₂, 300mM KC1), 16 nmol poly (dT) oligo (dA), 330pmol ³H-dGTP (specific activity 7.5°C103 cpm/pmol), 485 pmol ³H-dCTP (specific activity 5.2°C10³ cpm/pmol), 5.6nmol ¹⁴C-dATP (specific activity±60 cpm/pmol), crude extract containing approximately20μg of protein.
Incubation was carried out at 37°C for 30 min. Reactions were stopped by chilling and by adding 0.5ml of 0.1M sodium pyrophosphate and they were collected on a GC/G watman glass filter and washed with 5ml of 98% ethanol, dried, and the radioactivity was counted with liquid scintillation counter. The misincorporation frequency was expressed as the ratio of pmol of ³H-dGTP and ³H -dCTP: ¹⁴C-dATP incorporated into acid-precipitable materials. The misincorporation of DNA synthesis which used by tif 42°C (SOS⁺) extract was not increased than other cell free extract (SOS^-). For this reason, the enzyme activity of triphosphatase containd in cell free extract of Escherichia coli will rapidly hydrolyses dATP to dADP during incubation of reaction mixture.

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In pursuit of the cellular pathogenicity of periodontal disease, subcellular effects of bacterial endotoxin (lipopolysaccharide: LPS) on the tricarboxylic acid (TCA) cycle were studied using mitochondrial fractions from bovine periodontal ligament and dental pulp. Mitochondrial fractions were incubated with succinate-1, 4-¹⁴C as radioactive substrate for analyses of organic and amino acids synthesized both with and without LPS.
Addition of LPS caused obvious increase in amount synthesized, compared to control. Production of fumarate, α-ketoglutarate and malate increased 184, 330 and 362 percent, respectively, with addition of 100μg/ml E. coli 0127: B8 LPS.
Glutamate, glycine, alanine and aspartate were the major amino acids synthesized from bovine periodontal ligament mitochondrial fraction. Production of glutamate increased 148, 174and133 percent with addition of 50, 100 and 200μg/ml LPS, respectively.
These findings demonstrate that bacterial endotoxin can affect the process of periodontal disease by altering the metabolism and biosynthesis in mitochondria of the fibroblast.

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Matrix vesicles (MVs) in thin sections, were classified into four types: electronopaque MVs, lucid MVs, MVs of medium opacity, and MVs containing needle-shaped inclusions. In freeze-fracture replicas, they were classified into three types by the number of membrane-associated particles (MAPs) on the convex P-faces: MAP-rich MVs, MAPpoor MVs, and MVs with a moderate amount of MAPs. In the course of predentin maturation, electron-opaque MVs and MAP-rich MVs increased in number. It is believed that the increase of these MVs is necessary for the onset of mineralization.

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The antigenic relationship between amelogenins from developing rat enamel and carbonic anhydrase (CA) from rat erythrocytes was investigated by using a double immunodiffusion technique. Amelogenins ranging in size from 23, 000 to 30, 000 daltons formed a single precipitin line with an antibody against CA isoenzyme C. The results suggest that most of the amelogenins are derived from CA isoenzyme C.

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Localization of carbonic anhydrase (CA) in the developing dentin and enamel of the rat lower incisor was investigated by means of a histochemical technique at the electron microscope level. The CA reaction products were always localized in the area where initial mineralization i.e., nucleation of crystals occur in the developing dentin as well as enamel. In the enamel, CA activity was observed only in the initial marginal zone (10-20 μm in thickness) of the enamel at the matrix formation stage. The precipitates produced by the CA reaction appear in close proximity to the newly formed ribbon-shaped crystallites. CA activity in the dentin occurs in the calcification front (dentin-predentin border) of a few μm in thickness. Furthermore, the precipitates which appear on the predentin side are enclosed in the mass of initial crystals.
The present study confirms our previous observations at the light microscope level.

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