Carbamazepine 10,11-epoxidation in human liver microsomes: influence of the CYP3A5*3 polymorphism.

Carbamazepine (CBZ) is a commonly prescribed antiepileptic drug, and is mainly metabolized to 10,11-CBZ epoxide in humans. Its biotransformation is catalyzed by cytochrome P450 (CYP) enzymes, with the predominant isoforms being CYP3A4 and CYP3A5. In the present study, the effects of the CYP3A5*3 (rs776746) polymorphism on CBZ 10,11-epoxidation in human liver microsomes genotyped as CYP3A5*3 were examined using a kinetic analysis. The kinetics for CBZ 10,11-epoxidation fit the Hill model with n of approximately 1.9-2.1 in all liver microsomes of the wild-type (CYP3A5*1/*1) and heterozygous (CYP3A5*1/*3) and homozygous (CYP3A5*3/*3) variants. The S50, Vmax, and CLmax values of wild-type liver microsomes were 263-327 µM, 793-1590 pmol/min/mg protein, and 1.51-2.95 µL/min/mg protein, respectively. The Vmax and CLmax values of liver microsomes of the heterozygous variant were approximately 15-40% those of wild-type liver microsomes. On the other hand, the Vmax and CLmax values of liver microsomes of the homozygous variant were more similar to those of the wild-type than the heterozygous variant. These results suggest that the CYP3A5*3 polymorphism has a negligible effect on CBZ 10,11-epoxidation in an in vitro system using human liver microsomes.

Three dimensional architecture of lymphatic vessels in the tongue.

We have three-dimensionally reconstructed the lymphatic architecture of the rabbit tongue using a computer graphic three-dimensional reconstruction method together with histochemically stained serial cryo-sections. Three collecting lymphatic vessels lying in an anteroposterior orientation were identified in the tongue body. A superior longitudinal muscle accompanying collecting lymphatic (SLCL) that lies in the border between superior longitudinal muscle and transverse muscle was identified in this study, in addition to collecting lymphatics in the lingual septum (LSCL) and deep lingual artery accompanying collecting lymphatics (DLCL) that we reported previously. The vertical muscle accompanying collecting lymphatics (VCL) and the transverse muscle accompanying collecting lymphatics (TCL) were also identified as collecting lymphatics that joined the above three collecting lymphatics. Specific regional lymph flow was identified for each collecting lymphatic. A transverse right/left cross flow was identified for the LSCL, while the SLCL served the upper 1/3 of the tongue and the DLCL served the lower 2/3 of the tongue. Each collecting lymphatic that accompanied the internal lingual muscles joined to the SLCL, DLCL and LSCL, and were derived from blind-ended lymphatic capillaries that ran in the endomysium. Internal lingual muscle accompanying collecting lymphatics joined each other in the endomysium, and their blind-ended lymphatic capillaries had no branches.

Honeycomb structure in the lamina lucida of epidermal basement membrane during metamorphosis of Rana temporaria ornativentris.

In this study we examine the structure of the lamina lucida during metamorphosis of Rana temporaria ornativentris. During the metamorphosis of anuran larvae, both the epidermal cells and the dermal connective tissues in the tail regenerate. The basal surface of the epidermis becomes irregular and the epidermal basement membrane detaches from the epidermal cells, showing a widened lamina lucida. In this widened lamina we observed a geometrical honeycomb structure and a ladder structure. Each side of the honeycomb structure was approximately 40 nm and the intervals of the ladder structure were approximately 50 nm. From our observations we believe that the honeycomb and ladder appearances are different aspects of the same structure. At the beginning of metamorphosis anchoring filaments were prominent in the lamina lucida and, when the lamina lucida was tangentially cut, the lamina lucida showed the honeycomb structure. These results suggest that both the honeycomb and the ladder structures observed in the widened lamina lucida originate from constituents of the lamina lucida and become morphologically evident during the epidermal-dermal separation.

Degeneration and regeneration of the lip mucosal epithelium after cryo treatment in mice.

The process of degeneration and regeneration of the lip mucosal epithelium after cryo treatment was observed by transmission electron microscopy. The epithelial cells were degenerated by the formation of ice crystals and subsequently detached from the basement membrane, forming a blister cavity. The separation occurred between the epithelial cells and the lamina densa, leaving a small amount of cell debris on the lamina densa. The surviving cells at the periphery of the blister cavity, especially the cells in the basal half of the epithelium, provided the regeneration cells. They migrated over the cell debris, attached to the lamina densa and gradually phagocytozed it. Finally, they formed hemidesmosomes with the old lamina densa. The connections between the epithelial cells by desmosomes were so tight that desmosomes were preserved even between dead cells and between dead and living cells. Regenerating cells were moving in a multilayered form, remaining connected to each other by the dosmosomes. They were seen to divide by mitosis and thereby increase the number of the cell layer, whilst maintaining their connections with the neighbouring cells.

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium: an attempt to reactivate the 5'-nucleotidase activity with Mg++ ion supply.

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium was performed on cryostat sections using the 5'-nucleotidase (5'-Nase) staining method by light microscopy and backscattered electron imaging of scanning electron microscopy. The inhibition of the 5'-Nase activity by decalcification was cancelled by the Mg++ ion supply. The reaction products of 5'-Nase activity were produced on the lymphatic endothelial cells and the tubular structures of lymphatic vessels were seen as highlights by backscattered electron imaging. The invasion of 5'-Nase-positive lymphatic vessels into the alveolar bone from the periodontium was found in the present study.

Improvement of macromolecular clearance via lymph flow in hamster gingiva by topical warming and massage.

The lymphatic system is very important for macromolecular clearance in various tissues, especially in the gingiva. However, the kinetics of macromolecular clearance via the lymph flow in the gingiva are poorly understood. The aim of this study was to investigate whether thermal or mechanical stimulation affects macromolecular clearance via the lymph flow in the gingiva. Carbon black suspension was injected into the mandibular gingiva of anesthetized hamsters and its drainage into cervical lymph nodes was examined. Clearance of 14C-methylated bovine albumin and tritiated water from the gingiva and their drainage into submandibular lymph nodes and blood was quantified. The effect of topical warming or massage on clearance of 14C-methylated albumin from the gingiva during a 15 min period was examined. In addition, the influence of neurochemical antagonists on the stimulatory effect of topical warming on albumin clearance was investigated. Submandibular lymph nodes were clearly delineated by carbon black 10 min after the injection. More radiolabeled albumin appeared in submandibular lymph nodes than in serum, while more tritiated water appeared in serum. Topical warming (45 degrees C, 2 min) and warming plus massage (with a silicon rubber brush, 20 s) decreased the radiolabeled albumin in the gingiva 15 min after the injection. There was less radiolabeled albumin in the gingiva after gingival warming plus massage than after warming. Previous injection of HOE140 or propranolol into the gingiva diminished the stimulatory effect of topical warming on albumin clearance. It was concluded that topical warming plus massage improves macromolecular clearance via the lymph flow in hamster gingiva.

Regeneration of mouse lip epidermis after cryo treatment. Hemidesmosome formation and HSPG (heparan sulfate proteoglycan) distribution in basement membrane.

The processes of degeneration and the regeneration of the lip epidermal cells was observed by electron microscopy, focussing on the substance and the structure of the lamina lucida, on which regenerating cells migrated. After the repetitive freezing and thawing treatment, epidermal cells degenerated and detached from the dermis. The separation occurred between the epidermal cells and the basement membrane, leaving a small amount of cell debris on the lamina densa. After the separation of the epidermis, there were some thick parts in the lamina densa which appeared to be the part below hemidesmosomes. Regenerating epidermal cells migrated from the nondegenerated area along the cellular surface of the old lamina densa. They migrated over the cell debris which was gradually phagocitized, and formed new hemidesmosomes with the old lamina densa. Regenerating epidermal cells did not make close contact with the old lamina densa during their migration, but there was a clear space in between, indicating that some of the materials and the structure of the lamina lucida of the old basement membrane was preserved. By immunoelectron microscopy using anti-HSPG (heparan sulfate proteoglycan) antibody, it became clear that after the epidermal separation, HSPG was preserved in the basement membrane to some extent, especially in the thick parts of the lamina densa located below. The immunoelectron micrographs support the view that hemidesmosomes may reform at the previous locations at the old lamina densa.

Distribution of type VII collagen in the epithelial basement membranes of mouse palate, tongue and lip mucosa.

Dithiothreitol effectively separated the laminae densae and the laminae fibroreticulares of the basement membranes of oral mucosal epithelia as it does the epidermis and dermis. Dithiothreitol-separated epithelial basement membranes of palate, tongue, and lip were immunoreacted with colloidal gold-conjugated anti-type VII collagen antibody. By transmission electron microscopy, gold particles were observed only on the anchoring fibrils. For three-dimensional observation of the distribution of type VII collagen by scanning electron microscopy, secondary and backscattered electron images were compared. The secondary image showed the fine structure of the laminae densae and anchoring fibrils, and the backscattered images showed the gold particles conjugated with the antibody. By using an osmium conductive metal coating under optimal conditions, secondary and backscattered electron images of sufficient quality could be obtained. With the osmium coating, the backscattered electron image could show not only the gold particles but also the general morphological outline, making possible a comparison of the two images, which revealed the three-dimensional distribution of type VII collagen. Type VII collagen was also seen only on the anchoring fibrils, as in the epidermal basement membrane.

The advantage of the osmium conductive metal coating for the detection of the colloidal gold-conjugated antibody by SEM.

For the detection of the colloidal gold-conjugated antibody by scanning electron microscopy, the comparison between the secondary and the back-scattered electron images is necessary. After the various durations of osmium conductive metal coatings, enlargements of the diameters of the antibodies were observed. The thick coating reduced the contrast of the backscattered electron images. By the optimal thickness of the coating, the secondary electron image showed a minimum reduction in the resolution, and the backscattered electron image showed not only the gold particles with high contrast, but also the outline of the basic morphology.

Scanning electron microscopic observation of the epidermal basement membrane with osmium conductive metal coating.

Osmium conductive metal coating was performed for the scanning electron microscopic observation of the epidermal basement membrane. An extremely thin osmium coating induced the equivalent electrical conductivity as that of a thick platinum coating. As a result, by this coating, the resolution was only nominally reduced. The meshwork structure of the lamina densa and the three-dimensional morphology of the anchoring fibrils were observed in detail. Scanning electron microscopic observation of the osmium-coated specimen coincided with that of the transmission electron microscopic observations. It was evident that the osmium conductive metal coating is much more advantageous than the other coatings for obtaining high resolution images on the observations of the fine structures, such as the epidermal basement membrane.

Bifid ribs observed in the third and the fourth ribs.

Three cases of bifid ribs were found in two cadavers during routine dissections at the Iwate Medical University School of Dentistry. All of the cases were found in the third or the fourth rib. The distal parts of the osseous rib bifurcated with an angle of 60 degrees and both of the branches had their own costal cartilage. The costal cartilage fused again to form the trunk which was connected to the sternum. The space between the two branches was filled with presumably normal intercostal muscles. Blood supply was maintained by a small branch from the interthoracic artery to the upper branches. However, the intercostal nerves did not branch toward the upper branch but only ran along the lower margins of the lower branches of the bifid ribs.

Studies on the development of the articular part of the temporal bone with special reference to the postglenoid process.

The development and significance of the postglenoid process on the articular surface of the temporal bone were studied using 217 Indian skulls, which were divided by their level of tooth eruption into 6 developmental stages; preeruption period (Stage I), initial stage (m1 eruption) of the deciduous dentition period (Stage II), middle stage (m2 eruption) of the deciduous dentition period (Stage III), late state (eruption of 20 deciduous teeth) of the deciduous dentition period (Stage IV), mixed dentition period (Stage V), and permanent dentition period (Stage VI). Each skull was mounted on a Kraniophor in such a way that the auriculoorbital (Frankfurt) plane was positioned vertical to the horizontal plane and photographed at a focal distance of 40 cm. Using the Frankfurt plane as a base line, the vertical distances to the lowest point of the postglenoid process (a-A), to the deepest point of the mandibular fossa (b-B), and to the mid-point of the articular tubercle (c-C) were measured. The distances from various points of the mandibular fossa to the articular surface of the temporal bone were measured at the level of the Frankfurt plane. The results may be summarized as follows: The length of the postglenoid process as measured from the Frankfurt plane was 0.58 mm at Stage I and 0.75 mm at Stage II. It increased by 0.6-1.0 mm at each developmental stage. By Stage VI, it had increased to 3.85 mm (six- to seven-fold increase from the initial value). The anteroposterior width of the postglenoid process remained almost unchanged throughout the developmental stages. The growth of the articular structures of the temporal bone from the preeruption to deciduous dentition period increased 73% in the total anteroposterior dimension (5.22 mm) and 44% in the total vertical dimension (3.32 mm). The vertical dimension of the mandibular fossa began to rapidly increase following the eruption of the deciduous first molars.

Fine structure of the epidermal basement membrane of the lip: applications of dithiothreitol separation and ultrathin frozen sectioning.

The fine structure of the epidermal basement membrane at the electron-microscopic level has already been defined. To obtain more details, two techniques, dithiothreitol separation and ultrathin frozen sectioning, were applied either alone or in combination. Negatively stained ultrathin sections showed a much thicker lamina densa than ordinary plastic-embedded sections. In the lamina lucida, bridging filaments and subbasal dense plates were observed in negative images. After the treatment with dithiothreitol, the lamina densa could be peeled off mechanically from the underlying dermis, and the anchoring fibrils were pulled off the dermis, preserving the connection with the lamina densa. With this specimen, bundles of anchoring fibrils were observed clearly and their lengths could be measured. Negatively stained ultrathin sections of dithiothreitol-separated specimens showed the fine structure of the lamina lucida. Bridging filaments in the lamina lucida were resolved by negative staining.

New cavity forms in second deciduous molars which maximize dentin thickness between cavity and pulp chamber.

This study was designed to devise configuratively ideal cavity formations using 49 second deciduous molars, consisting of 25 extracted from the maxilla and 24 from the mandible. A cavity having at least 1 mm subcavitary (below the cavity) dentin thickness at any point of measurement and having an appropriately formed and positioned retention was defined as an ideal cavity. The same methods were used as in our previous study of first deciduous molars. The following results were obtained: 1. In upper second deciduous molars, the subcavitary dentin thickness was thin at the mesiobuccal side of the cavity; it measured 0.9 mm. In lower second deciduous molars, the subcavitary dentin thickness was thin at the mesiobuccal, distolingual sides of the cavity, and central fossa, where the retentional groove was provided; it measured 0.8-0.9 mm. The other measured values exceeded 1.0 mm. All thickness measurements were very close to the values considered as ideal. 2. In upper second deciduous molars, the margins of the cavity were positioned medially to the summits of respective cusps, 1.8-2.0 mm at the buccal side and 1.5 mm at the lingual side. The entire cavity was located lingually. 3. In lower second deciduous molars, the buccal margin of the cavity was positioned 1.7 mm medially to the summit of the distobuccal cusp and 1.2-1.3 mm medially to the summits of the other buccal cusps. The lingual margin was positioned 1.4-1.5 mm medially to the summits of respective cusps. medially to the summits of respective cusps. 4. The buccolingual width of the cavity amounted to 1/3 of the distance between the summits of the buccal and lingual cusps in both upper and lower second deciduous molars. 5. At the mesial side, the depth of cavity was 1.4 mm at the buccal wall and 1.5 mm at the lingual wall in upper second deciduous molars, and 1.4 mm at the buccal wall and 1.6 mm at the lingual wall in lower second deciduous molars. 6. The width of gingival wall in the proximal box measured 1.0 mm in both upper and lower second deciduous molars.

New cavity forms in first deciduous molars which maximize dentin thickness between cavity and pulp chamber.

Using a total of 60 human extracted first deciduous molars (30 upper molars and 30 lower molars), we contrived a variety of ideal cavities having dentin thicknesses below the cavity (subcavitary dentin) that measure approximately 1 mm in thickness from the pulp chamber at any point of measurement and also having a retentional groove prepared in such a way that detachment of a filling material is prevented. The transparent specimens prepared from the cavity-containing teeth were cut into serial sections of 93 microns. These sections were reconstructed using a personal computer. The thickness of dentin below the cavity was measured in randomly selected sections. Results obtained were as follows: 1. An ideal thickness of the subcavitary dentin was preserved for upper first deciduous molar by preparing a retentional groove lingually or by providing a dovetail-like shape to mesio-distolingual sides in the cavity; for lower first deciduous molar, any form of cavitation worked. 2. In the upper first deciduous molars, the margins of the cavity were displaced medially to the summits of the respective cusps 1.9 mm at the buccal side and 1.2 mm at the lingual side. In the lower first deciduous molars, the buccal margin medially measured 0.9 mm and the lingual margin measured 0.6 mm at the mesial side. At the distal side, the buccal and lingual margins measured 1.2 mm and 1.0 mm, respectively. 3. The ratio of cavity width to the distance between the summits of the buccal and lingual cusps was 1/3 in upper deciduous first molars and 2/5 in the lower first deciduous molars. 4. In the upper first deciduous molars, the depths of the buccal and lingual walls of the cavity at the center of the central groove were both 1.1 mm. In the lower first deciduous molars, the cavity formed with its center at the middle of the transverse ridge had a depth of 1.5 mm at the buccal wall and a depth of 1.7 mm at the lingual wall. The cavity formed with its center at the central fossa had a depth of 1.2 mm at the buccal wall and 1.1 mm at the lingual wall. 5. The width of the gingival wall in the proximal box measured 0.6-0.7 mm in upper and lower first deciduous molars.

[Morphological studies on the margo infraorbitalis (on the longitudinal changes of infraorbital suture)].

The purpose of this study was to obtain information on the developmental changes of the infraorbital margin and the form of infraorbital suture. 136 Indian craniums were divided into five developmental groups according to the stage of eruption of the teeth. The anterior view of the skull was observed photographically. The results are summarized as follows: 1. The infraorbital margins were divided into three types according to the shape of the infraorbital suture. Type I: Infraorbital suture was independent. Type II: Infraorbital suture and zygomatico-facial suture coincided. Type III: Infraorbital suture and zygomatico-facial suture were joined. Type I was the basic type of infraorbital suture. 2. The frequency of type I was 90% at the pre-eruption stage, 31.8% at the eruption stage, 41.7% at the deciduous dentition stage, 56.3% at the mixed dentition stage, and 62% at the permanent dentition stage. 3. On the distance between the median line and each point of the infraorbital margin the growth rate is expressed in values relative to a standard that was based on the pre-eruption stage. The growth rate was greatest at the highest point of the superior margin in the infraorbital foramen (1.79 times), the middle rate was at the position of the infraorbital suture (1.72 times) and the smallest rate was 1.63 times at the position of the zygomatico-facial suture. Each measuring point moved towards lateral side.

[Longitudinal changes in amounts of maxillary growth].

The purpose of this study is to investigate postnatal developmental changes in the shape of the maxilla. Normal maxillary form at each development stage and amounts of growth were analyzed by longitudinally measuring 136 Indian skulls. Maxillary growth changes were assumed on the basis of mean values and growth rates of each region measured. Results 1) Vertical growth Vertical growth of the maxilla (N-Pr) at the fifth (permanent-dentition) stage approximately doubled growth in the first (pre-eruption) stage. The corpus of the maxilla and its alveolar process lay beneath the foramen infraorbit. Vertical growth in this region (m-Pr) increases rapidly during the period of eruption of deciduous and permanent teeth. In permanent dentition, the vertical dimensions of the upper (N-m) and lower (m-Pr) parts of the foramen infraorbit were almost the same. 2) Growth in width As the eyes grow, the upper face widens. Resulting development in the tensed zygomatic-maxillary suture at the inferior margin of the orbit expands the upper surface of maxilla. Maxillary width increased by approximately 1.8 times between the first and fifth stages.