Angiotensin-I converting enzyme inhibitory peptide derived from porcine skeletal muscle myosin and its antihypertensive activity in spontaneously hypertensive rats.

Crude myosin light chain was extracted from Japanese domestic pork loin and digested with pepsin. Antihypertensive peptide was isolated from this digest as a measure of its inhibitory activity for angiotensin-I converting enzyme (ACE). Through isolation with some chromatographies, a single active fraction was isolated, and it was detected as an octapeptide, Val-Lys-Lys-Val-Leu-Gly-Asn-Pro, from 47th to 54th positions of myosin light chain. The 50% inhibitory concentration of this peptide was 28.5 muM. Kinetic evaluation showed that this peptide was a noncompetitive inhibitor, but it was slowly hydrolyzed by ACE. At the dose of 10 mg/kg, this peptide showed antihypertensive activity after a maximum of 3 h of administration and was estimated as a temporally effective hypotensor.

Histological studies on the effects of tooth brushing on repair of alveolar bone after periodontal osseous surgery in the rat incisor.

The purpose of this study was to elucidate the effects of tooth brushing on repair of alveolar bone after periodontal osseous surgery in the labial alveolar bone of rat incisor. The surgery was performed on 24 Wistar rats divided into 2 groups: the experimental group, which was subjected to tooth brushing, and the control group, which was not. In the experimental group, daily tooth brushing was initiated at 4 weeks after surgery. The rats were sacrificed after 1 or 2 weeks of tooth brushing. Microradiographic, light and fluorescence microscopic examinations were made of sections of the alveolar bone and its surrounding tissue. After 1 week of tooth brushing, callus with a low degree of mineralization and with large, irregularly arranged, young osteocytes appeared in the superficial layer and crest portion of alveolar bone in the brushing region. Numerous blood vessels had invaded the callus. In this region, the height of osteoblasts on the callus surface increased. At the alveolar crest region, the callus was approximately 3 times thicker than in the superficial region. After 2 weeks of tooth brushing, modification had occurred in the callus; this region had evolved into developed bone with a compact matrix. These findings suggest that the intermittent mechanical stress of tooth brushing is useful in activating the cells of the alveolar periosteum and in stimulating bone formation.

Electron microscopic study of the cytoskeleton of osteoblasts in rat alveolar bone: microfilaments and intermediate filaments as demonstrated by detergent perfusion.

To visualize the cytoskeleton and to preserve the morphology of osteoblasts in the alveolar bone, rats were perfused with a mixture of Triton X-100 and a low concentration of glutaraldehyde, and their alveoli were observed under a transmission electron microscope. The alveolar bone osteoblasts contained a large number of fibers (5-6 nm in diameter) which seemed to be microfilaments. In the cytoplasm just below the cell membranes at the osteal cell surfaces, the microfilaments formed thick bundles distributed in parallel with the long axis of the cell. These findings suggest that, in rat alveolar bone osteoblasts, the bundles of microfilaments located immediately below the cell membranes at the osteal cell surfaces are stress fibers in situ.

Cytoskeletal organization in fibroblasts of rat calvaria periosteum: microfilaments demonstrated by detergent perfusion.

The authors examined the microfilament of fibroblasts in rat calvarial periosteum by transmission electron microscopy after perfusing rats with a mixed solution of Triton X-100 and a low concentration of glutaraldehyde. Fibroblasts in the periosteum were fusiform and had elongated oval nuclei. Microfilaments with diameters of 5-6 nm were noted within the cytoplasm. Although these microfilaments were parallel to the long axis of the cell, stress fibers, which are thick bundles of microfilaments present in cultured fibroblasts, were not observed in these fibroblasts.

Scanning electron microscopic observation on the dentin surface of rat enamel-free areas.

Formation of the enamel covering on the dentin of the tooth crown is thought to depend upon the dentin surface structure during the early stages of enamel development. For this reason, observations were made of the dentin surfaces of both the enamel-free area (EFA) and the region beneath the enamel area. After treatment with sodium hypochlorite and formic acid, scanning electron microscope observations were made of the EFA surface and the exposed dentin surface beneath the enamel of unerupted rat mandibular first molars. Cone-like projections consisting of matrix fibers were observed on the dentin surface covered with the enamel. These cone-like projections formed continuous ridges that connected with the network structure. At the concavities of these network structures these projections were distributed sparsely. Instead of the cone-like projections, the matrix fibers formed a coarse network structure along the surface at the EFA.

Immunohistochemical study of the cytoskeleton of osteoblasts in the rat calvaria. Intermediate filaments and microfilaments as demonstrated by detergent perfusion.

We evaluated the cytoskeleton of osteoblasts in the rat calvaria by transmission electron microscopy after perfusion with a mixture of Triton X-100 and glutaraldehyde and immunoelectron microscopy. In osteoblasts, microfilaments (5-6 nm in diameter) and intermediate filaments (10-12 nm) were arranged in networks or bundles, avoiding cell organelles such as mitochondria and the rough-surfaced endoplasmic reticulum. Some of these intermediate filaments were attached to the nucleus and had a double-helix structure. Immunoelectron microscopy demonstrated that these fibers are actin and vimentin filaments, respectively. Intermediate filaments were abundant around the nucleus while microfilaments were abundant around the cell membrane. Microfilaments formed thick bundles around the cell membrane in formative osteoblasts but did not form bundles in resting osteoblasts.

Organic structures of the hypercalcified peritubular matrix in horse dentine.

EDTA-insoluble organic structures of the hypercalcified peritubular matrix (PM) in horse dentine were observed by scanning electron microscopy. The PM was enveloped in double cylindrical structures composed of fibrillar sheaths in the inner and outer peripheries. Between the outer fibrillar sheath and intrinsic fibrils of the intertubular matrix, a calcified cementing membrane existed. Within the PM, warped cone-shaped structures of fibrillar sheaths, overlapping at intervals of 4-6 microns and semiconcentrically surrounding the dentinal tubule, extended from the inner fibrillar towards the outer fibrillar sheath. The cone-shaped fibrillar sheaths following the inner and outer fibrillar sheaths were identified as the incremental lines of the PM. Most of these fibrils may be collagen although it could not be confirmed, whereas non-collagenous organic materials in the lateral branches of the dentinal tubule are radially arranged in the PM. These EDTA-insoluble structures were three-dimensionally illustrated using an image-analysing system.

Inorganic components and the fine structures of marginal and deep subgingival calculus attached to human teeth.

Inorganic components and the fine structures of marginal ledge-type and deep subgingival spiny deposits in human old dental calculus were investigated by scanning electron microscopy and energy dispersive electron-probe microanalysis. The ledge-type deposits consisted of the extra- and intracellular calcifying deposits, large plate-shaped crystals, and bacillus-shaped deposits composed of hexahedrally based crystals. The spiny deposits were mainly formed by aggregations of the bacillus-shaped deposits. In the outer and middle layers of the spiny deposits, the Ca, P, and Mg concentrations were all significantly higher than those of the ledge-type deposits. A consideration of the crystal shapes and Ca, P, and Mg molar ratios reveals the following differences. Calculus components of the ledge-type deposits contained crystal types quite similar to sandy grain-shaped hydroxyapatite (HAP), plate-shaped octacalcium phosphate (OCP), and hexahedral Mg-containing whitlockite (WHT). On the other hand, in the spiny deposits, the Mg-containing WHT type comprised a large proportion of the calculus; the HAP type was found in the outermost and inner layers; and no OCP type was detected.

The expression of actin mRNA in megakaryocytes demonstrated in paraffin sections, and epoxy resin semi-thin and thin sections by in situ hybridization.

Megakaryocytes of dog bone marrow were utilized as target cells for identifying actin mRNA expressing cells on semi-thin and thin sections. After in situ hybridization with radioisotope-labeled probes was performed on paraffin sections, gelatin capsules containing freshly prepared epoxy resin were placed on the sections. The resin was solidified and detached from the slide glass, and semi-thin and thin sections were obtained. The signals showing actin mRNA expression were detected on megakaryocytes in these sections by light and electron microscopy.

Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of the pig and rat.

Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13-17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149-173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1-148 of the antigen. Immunoelectro-transfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13-17 kDa nonamelogenins. The antiserum against the 13-17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13-17 kDa nonamelogenins.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional observations of the relation between the natural enamel surface and the outermost layer in human permanent tooth.

A coronal enamel surface of a human permanent tooth and the exposed outermost layer etched with phosphoric acid or EDTA were observed in the same field by means of scanning electron microscopy. Gross structural patterns of the enamel surface were either of a flattened type showing no perikymata patterns of of undulating and overlapping types with grooves and convex ridges. The undulating and overlapping patterns showed an alternating structure of rows of numerous shallow and deeper prism-end pits or that of these pit rows combined with a smooth band. The pit rows were usually located in the grooves with succeeding slopes in the middle of coronal enamel, but, towards a cusp, they often existed in the convex ridges. The striae of Retzius reached the grooves of perikymata from cervical to the middle of coronal enamel; however, the arrangement of the striae did not always follow perikymata patterns towards a cusp. The overlapping pattern in cervical enamel and the flattened type in cuspal and cervical enamel usually had uniform structures of shallow, indistinct, or no prism-end pits. The surface 'prismless' layers containing indistinct prism structures were observed under these uniform structures.

Scanning electron microscopic observations of microcanals and continuous zones of interglobular dentin in human deciduous incisal dentin.

Scanning electron microscopic (SEM) observation of the coronal dentin of deciduous incisors revealed microcanals and continuous zones of interglobular dentin (CID) in the labio-lingual central portion of dentin beneath the incisal edge. The microcanals, which were clearly larger than dentinal tubules, extended from the incisal edge to the vicinity of the pulp cavity. They were located essentially in the labio-lingual central portion of the dentin and were arranged linearly in the mesio-distal direction. Inside the microcanals, collagen fiber bundles were arranged almost in parallel with the canal long axis: spherical bodies 1.0-2.5 microns in size made up of assemblies of regular parallelpipedal crystals and granulated crystals were also seen. In some instances, bacteria had invaded incisal dentin that had been exposed by attrition. The CID were made up of interglobular dentin aligned with the long axis of the tooth. As was true in the case of the microcanals, several zones were arranged irregularly in the mesio-distal direction, generally in the labio-lingual central portion of the coronal dentin. The CID were confined to about half the width of the incisal edge dentin on the incisal edge side and did not appear in the vicinity of the pulp cavity. Within the interglobular dentin itself, which was surrounded by calcospherites, were longitudinal collagen fibers connected with dentinal tubule walls inside calcified dentin and collagen bundles forming a network with those fibers. Spherical bodies 1.0-1.5 microns in size were observed attached to the surfaces of the bundles or distributed among these fibers. In some instances, the microcanals penetrated the CID.

Morphological changes in human and animal enamel rods with heating--especially limits in temperature allowing discrimination between human and animal teeth.

Little has been revealed about making discrimination between human and animal using a piece of tooth found in a burned cadaver. From the viewpoint of forensic dental medicine, it is a theme no less valuable. This experimental study was attempted for this reason. Teeth from an individual body of human, monkey, dog, rabbit and rat were heated in turn on the muffle furnace. The heating temperatures were from 200 degrees C to 1,000 degrees C in time spans of 5, 30 and 60 minutes. After heating, each tooth and its control were observed by a scanning electron microscope (magnifying power: 3,500 x or 3,600 x). At heating temperature of 500 degrees C or 600 degrees C, enamel starts to come off in lumps and cracks appear in the enamel rods. The arcaded form in human and monkey, hexagon in dog, elongated chain in rabbit, rows of short, diagonal parallel lines equally directed at every other row in rat--these basic morphological features of the enamel rods--are retained till the heat reaches 700 degrees C. The enamel rods in monkey yield to heat more easily than those in human. At 600 degrees C many cracks appear and deformation of the arcaded form starts. With heating of 5 minutes at 800 degrees C the outline of the pattern is obscured. Human and animal teeth get varied forms of cracks in the enamel rods with heating more than 5 minutes at 800 degrees C. The structure of the enamel rods is broken and morphological characteristics are lost. This makes discrimination of human and animal quite difficult.(ABSTRACT TRUNCATED AT 250 WORDS)

[Ultrastructural and cytochemical studies of clonal cell lines in fibroblasts derived from human periodontal ligaments--establishment of clonal cell lines in fibroblasts from periodontal ligaments].

It has recently been suggested that fibroblasts in periodontal ligaments have cytochemical characteristics similar to those of osteoblasts and different from those of other connective tissues. The authors isolated clonal cell lines of fibroblast-like cells from human periodontal ligaments in order to clarify their nature. Digestion with collagenase and hyaluronidase was used to isolate the cells from a human periodontal ligament. The cells were then plated in a 96-hole microplate. A single cell in a conditioned medium containing 20%FBS was placed in each hole. From these single cells large colonies ware subcultured. Subculturing was done every 8 days until more than 20 successive generations had been produced. The method developed by Lowry et al. was used to determine the ALPase activity of the cultured cells. From the 768 cells cultured from human periodontal ligament, 7 clonal cell lines were isolated in vitro. Cultures of these clonal cell lines resulted in typical, spindle-shaped, fibroblast-like cells, all of which were homogeneous. Very high ALPase activity was observed in 4 of the 7 cell lines. Enzyme reaction products occurred mainly along cell membranes. These stable clonal cell lines provide suitable systems for in vitro studies related to morphological and functional analysis of fibroblasts in the periodontal ligament.

[Morphological study of responses to mechanical stretching in fibroblasts derived from human periodontal ligaments].

Clonal cell lines established from human periodontal ligaments were used in vitro experiments to ascertain periodontal ligament responses, on the cellular level, to mechanical stretching. A procedure developed by Hasegawa et al. (1985) was used in applying mechanical stretching. Unstretched cultures were used as controls. All cultures were processed for investigations of fine structures, histochemical and cytochemical detection of ALPase activity, and localization of alpha-tubulin. Cultured cells to which mechanical stretching was intermittently applied showed little change in overall appearance, cell shape and arrangement, and distribution of alpha-tubulin. Although fine structural characteristics remained unchanged in both stretched and unstretched cultures, mechanical stretching force clearly increased the intensity of ALPase activity. Histochemical and cytochemical examinations indicated that the added intensity resulted from increases in numbers of cells demonstrating enzymatic activity.

Bacillus-shaped deposits composed of hexahedrally based crystals in human dental calculus.

In human supra- and subgingival calculus, bacillus-shaped deposits showing various rocky-pile forms composed of hexahedrally based crystals were observed by scanning electron microscopy. The crystal size measured approximately 0.1-1.5 microns. The electron probe microanalysis always detected calcium, phosphorous, and magnesium. Their molar ratios resembled those of magnesium-containing whitlockite and moreover the crystals also gave the electron diffraction pattern of whitlockite. The bacillus-shaped deposits happened to coexist with the intracellular calcifying microorganisms, furthermore, oral microorganisms partially replaced by the hexahedrally based crystals were found. The crystal deposits were never seen in the surface layers of calculus exposed to the oral cavity, but occurred in the innermost layers and intra-spaces of supragingival and ledge-type subgingival calculus and in the outer layers of deep subgingival calculus.