Comparison of posterior alveolar canal location measured on computer tomography scan with cadaveric measurement of posterior superior alveolar foramen in Japanese samples.

The aim of this study was to analyse anatomical characteristics of the most posterior alveolar canal (PAC) on computed tomography (CT) images and the posterior superior alveolar foramen (PSAF) physically identified in cadaveric samples, to avoid injuring the posterior superior alveolar artery (PSAA) during surgery in the maxillary tuberosity region. The study included 125 hemi-heads of 64 Japanese cadavers. Simple CT data of the maxillary bone region of the samples were obtained and analysed using measurement software. The alveolar crest (AC) and the PAC were identified to calculate the shortest distance between the AC and the PAC (AC-PAC). Then the samples were dissected to measure physically the shortest distance between the AC and the PSAF (AC-PSAF). The data were analysed statistically. The mean value and standard deviation were 20.7±4.2mm for AC-PAC and 20.7±4.3mm for AC-PSAF. The intraclass correlation coefficient between AC-PAC and AC-PSAF was 0.98. The CT-measured PAC locations were found to be almost identical to the PSAF positions identified physically in the samples. Preoperative CT localization of the PAC aids in avoiding injury to PSAA, while preoperative CT evaluation is important for each case due to significant individual variability in the anatomical PAC and PSAF locations.

Computed tomography and anatomical measurements of critical sites for endosseous implants in the pterygomaxillary region: a cadaveric study.

The aim of this study was to obtain computed tomography (CT) and physical measurements of the pterygomaxillary region to determine the anatomical and radiographic landmarks that clinicians need for pterygoid implant placement. Seventy-eight hemi-heads with an atrophic posterior maxilla from 46 cadaveric samples were measured using CT. Twenty-one hemi-heads were selected randomly for physical measurements. CT measurements showed that the mean and minimum distance between the maxillary tuberosity point (MT) and the most lateral lowest point of the pterygomaxillary fissure (PF) were 18.7mm and 10.0mm, respectively. The mean and minimum distance between the alveolar crest point passing the extended line of the infrazygomatic crest and the PF were 22.7mm and 14.7mm, respectively. The mean and minimum distance between the PF and the greater palatine canal were 2.9mm and 0.2mm, respectively. Physical measurements showed that the mean and minimum distances between the MT and the descending palatine artery (DPA) were 19.4mm and 12.7mm, respectively, and those between the PF and the DPA were 3.7mm and 0.0mm, respectively. The results confirmed considerable variation in the values of the pterygomaxillary region measured at the specific sites. Therefore, careful and sufficient consideration is required in each case of pterygoid implant placement.

Anatomical relationship between the sublingual fossa and the lateral lingual foramen.

This study investigated the locational relationship between the sublingual fossa (SF) and the lateral lingual foramen (LLF) in order to gain useful knowledge so that perforation of the lingual cortical bone and damage to the adjacent blood vessels can be avoided when placing an endosseous implant (implant) in the mandibular interforaminal region. The deepest point of the SF (SFP) and the LLF were identified in 38 Japanese cadaver mandibles (20 edentulous and 18 dentate) by computed tomography (CT) and physical measurement. Their locations were measured. In the edentulous cases, the SFP was located approximately 15 mm vertically from the alveolar crest in the direction of the mandibular lower margin in the canine and premolar regions, and the LLF was located within a 5mm radius from the SFP. Thus, significant attention to the locational relationship between the SFP and the LLF, as seen on preoperative CT, is required when placing an implant ≥3.75 mm in diameter and ≥15 mm in length in this region.

Sexual Dimorphism of Endocranial, Facial and Limb Measurements in the Yellow Baboon (Papio cynocephalus).

The present study focused on the sexual dimorphism of yellow baboon (Papio cynocephalus) to clarify its relationship with social behaviour. The degree of sexual dimorphism in the endocranial volume is the lowest among the investigated measurements. Among the facial measurements, the degree value of sexual dimorphism was the maximum (38.4%) for palate length and the minimum for palate breadth at the upper second molar (M2) (16.8%). Reduced major axis (RMA) regression analysis indicated that most positive allometry in relation to body mass was barely shown in the endocranial volume, palate length, palate breadth at M2 and mandibular ramal width. On the other hand, most negative allometry in relation to body mass was barely indicated in the bizygomatic breadth, skull length, humeral length and femoral length. The plate breadth at M2 in males was smaller than that in females in equivalent to body mass. The results of the present study suggest that more males have longer pointed muzzles than females, which is considered to create an impressive view of large canine teeth. This contributes to display among males and agonistic encounters rather than to necessity of increased facial size due to larger body size or dietary influences.

Anatomic measurement of the depth and location of the sublingual fossa.

The purpose of this study was to measure the depth and location of the sublingual fossa, a potential site of sublingual bleeding/lingual cortical perforation during endosseous implant placement in the mandibular interforaminal region (MIR), to clarify anatomical variation. Using the mandibles of 37 Japanese cadavers, the lingual depth (LD) between the lingual surface and the line perpendicular to the inferior margin of the mandible (IMM), as well as the vertical distance (VD) between the lingual surface and the IMM or the mental foramen (MF) level, were measured at defined points and lines within the MIR. The definite sublingual fossa (SF) was identified by the LD (≥ 1.0mm) and the VD, and the depth and location of the SF were determined. The depth ranged between 1.0mm and 5.8mm, and the vertical location ranged between 9.2mm and 15.7 mm from the IMM and between 2.2mm and 6.1mm from the MF level. These results revealed certain tendencies in the depth and location of the SF but the variation was substantial. The SF should be identified in each case as accurately as possible by CT before implant placement in the MIR to minimize the risk of the potential complications.

Differential expression of major gap junction proteins, connexins 26 and 32, in rat mammary glands during pregnancy and lactation.

We examined the expression pattern of two major gap junction proteins, connexin 26 (Cx26) and connexin 32 (Cx32), in rat mammary glands during pregnancy and lactation. Immunohistochemically the two different Cxs were coexpressed in acinar cells and were independently modulated according to the physiological cell activity. Western blot analysis demonstrated that Cx26 gradually increased from early pregnancy, while Cx32 rapidly and dramatically increased at 16 h after parturition, and that both Cxs reached a maximum early in lactation. Increased expression of both Cxs was confirmed by Northern blot analysis showing that their mRNA transcripts were significantly induced on the day of parturition. We also analyzed double-immunofluorescent staining for Cx26 and Cx32 on a confocal laser scanning microscope, in order to examine colocalization of these Cxs in situ. Cx26 immunoreactivity mostly overlapped with Cx32-positive sites in acinar cells of lactating mammary glands, indicating that both Cxs were colocalized together in the same gap junctional plaques in lactation. These results suggest that upregulation of Cx26 and Cx32 in acinar cells at lactating stages, with colocalization in the same gap junctional plaques, may be important for control of secretion by acinar cells in rat mammary glands.

Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice.

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition.

Heme oxygenase1 (HSP-32) is induced in myelin-phagocytosing Schwann cells of injured sciatic nerves in the rat.

Schwann cells participate in myelin phagocytosis in the early stage of Wallerian degeneration, prior to the recruitment of macrophages. This is the first report that Schwann cells induce heme oxygenase-1 (HO-1), a 32-kDa heat shock protein, only when they have transformed into myelin-phagocytosing cells from myelinating cells (days 2-3) immediately after crush injury of rat sciatic nerves. Double immunofluorescent labelling for HO-1 and transferrin receptors revealed that HO-1-immunoreactive Schwann cells also expressed transferrin receptors suggesting activation of iron metabolism. The transient induction of HO-1 in Schwann cells may contribute to the adaptive function in an altered environment when the cells have lost contact with axons, and may play a crucial role in the ensuing regeneration.

An improved method for avulsion of lumbar nerve roots as an experimental model of nitric oxide-mediated neuronal degeneration.

A root avulsion lesion on the spinal nerve of adult animals is a useful technique to make a model for axotomy-induced motoneuronal degeneration, which is thought to be mediated by nitric oxide (NO). Here, we show a simplified version of extravertebral avulsion in the young adult rat. The L4 nerve always runs under the transverse process of the L5 vertebra, which is located just rostral to the delineation of the iliac crest. We used the iliac crest as a clue for the identification of the L4 nerve during surgery, including before skin incision. In almost all animals the L4 nerve was successfully avulsed at the exit point from the spinal cord. This experimental result was similar to that shown in the previous literature; the number of either Nissl-stained or ChAT-immunoreactive (-ir) motoneurons (MN) gradually decreased, while NOS immunoreactivity was induced in the MN after avulsion. Furthermore, a combined method of confocal laser scanning microscopy and double fluorescent procedures carried out in this model suggested the existence of cellular interaction between NOS-ir MN and OX42-ir or ED1-ir microglia. It is concluded that this simple and fast method of spinal root avulsion is very useful for making a reproducible model of NO-mediated MN cell death, with which the mechanism of neuronal cell death, including neuron-glia interaction, can be further explored.

Changes in the distribution of peanut agglutinin (PNA) binding molecules during muscle reinnervation following nerve crush injury.

Peanut agglutinin (PNA) staining during muscle reinnervation following a crushing injury of the sciatic nerve was performed in reference to the neural profiles immunolabeled with the PGP 9.5 antibody. PNA staining in the normal controls exhibited dots, granules, or lines along the length of the nerve fibers in the nerve trunk, but was faint or absent in the motor endplate. At seven days post-crush, PNA staining was detected around the vacuolated neural structures in the disorganized nerve trunk, but was still faint or absent in the motor endplate. At twenty-one days post-crush, when PGP 9.5-positive regenerating axons appeared in most of the motor endplates, PNA staining, either faint or strong, followed the pathway of the nerve fibers delineated by PGP 9.5-like immunoreactivity. During reinnervation to the motor endplates, PNA staining displayed signs of remodeling in the nerve trunk, such as marked variations in density and profile in the nerve fiber-associated dots or patches; it increased in intensity in the connective tissue covering the area of the motor endplate, as well as in the junctional myofiber surface. The structures recognizable by PNA coincided with components of the connective tissue such as collagen fibers and capillaries. Results suggest that: 1) the expression of PNA-binding molecules is dependent on the state of innervation, and 2) the spatiotemporal relationship between neural profiles and PNA staining provides sequences of axonal extension and subsequent nerve terminal maturation during regeneration in the motor endplate.

Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes.

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.

Induction of apoptosis in maxillary sinus cancer cells by 5-fluorouracil, vitamin A and radiation (FAR) therapy.

The triple combination of 5-fluorouracil (5-FU), vitamin A and radiation (FAR therapy) has been used since 1972 to treat malignant tumors of the head and neck at Kyushu University. Using nick end labeling of tumor specimens, cells of human maxillary sinus carcinomas were observed previously to undergo apoptosis in response to FAR therapy. The present study evaluated the in vitro effects of FAR therapy on a human maxillary sinus cancer (IMC-4) cell line. We further compared the effects of FAR therapy on this cell line with those effects seen on tissue samples taken from patients with maxillary sinus cancers. DNA electrophoresis and electron microscopic examination of the IMC-4 cells after treatment with FAR therapy revealed typical apoptotic features. The effects of 50-100 micrograms/ml 5-FU, 10(-4) M all-trans-retinoic acid and radiation to 6 Gy on IMC-4 cells were evaluated by trypan blue dye exclusion and a cell colony formation assay. 5-FU and radiation caused direct cell death, while vitamin A mainly inhibited cell growth. The combination of these treatment as FAR therapy synergistically enhanced cell death and inhibited cell growth. Flow cytometry demonstrated that FAR-treated cells were arrested in the G1 phase of the cell cycle before undergoing apoptosis. To further investigate possible biological parameters influencing a tumor's apoptotic sensitivity, we also examined the expression of p53 in human maxillary sinus cancer cells and analyzed the relationship between p53 expression and apoptosis. However, no relationship was found between these two markers at the time point studied.

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon.

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement.

Expression of NOS, PSA-N-CAM and S100 protein in the granule cell migration pathway of the adult guinea pig forebrain.

To investigate the possible role of nitric oxide (NO) in adult neurogenesis and neuron-glial migration in the rostral migratory stream (RMS), we used a double-labeled immunofluorescence technique together with confocal laser scanning microscopy, and examined the localization of nitric oxide synthase (NOS), the highly polysialylated isoform of neural cell adhesion molecule (PSA-N-CAM), and the astroglial marker in brain, S100 protein (S100), throughout the length of the subependymal layer (SEL) to olfactory bulb (OB) pathway of the adult guinea pig forebrain. Blast-like, beaded, clustered immature cellular elements stained for PSA-N-CAM and those having a typical astrocytic phenotypes positive for S100 protein were densely interlaced throughout the entire length of the SEL. Some S100 positive ependymoglial cells (tanycytes) gave off their basal projections into the closely packed PSA-N-CAM immunopositive clusters in the rostral extension of the subependymal zone (SEZre). The SEL was devoid of NOS immunoreactivity. A dense network of punctate, fenestrated and radially oriented immature cellular elements positive both for NOS and PSA-N-CAM intermingled and overlapped in the inner part of the internal granular layer (IGr), whereas in the outer part, PSA-N-CAM expression gradually diminished and the cells shifted to mature bipolar, spherical or spindle-shaped granule cells with uniform cellular contours, which were exclusively immunopositive for NOS. Radially oriented astroglial phenotypes were intertwined with PSA-N-CAM neuronal clusters in the SEL, and were closely apposed to NOS neuronal elements in the IGr. In summary, these results showed a distinct separation of neurons and glia as revealed by PSA-N-CAM and S100 protein immunostaining, and an inverse spatio-temporal correlation of expression between PSA-N-CAM (immature neuroblasts) and NOS (mature neurons) in the adult guinea pig RMS.

Specific localization of gap junction protein, connexin45, in the deep muscular plexus of dog and rat small intestine.

Cellular networks of pacemaker activity in intestinal movements are still a matter of debate. Because gap-junctional intercellular communication in the intestinal wall may provide important clues for understanding regulatory mechanisms of intestinal movements, we have attempted to clarify the distribution patterns of three types of gap junction proteins. Using antibodies for connexin40, connexin43, connexin45, smooth muscle actin, and vimentin, immunocytochemical observations were made with the confocal laser scanning microscope on cryosections of fresh-frozen small intestine and colon of the dog and rat. Connexin 45 was localized along the deep muscular plexus of the small intestine in both dog and rat. Double labeling studies revealed that connexin45 overlapped with vimentin -, but not actin-positive areas, indicating the fibroblast-like nature of the cells, rather than their being smooth muscle-like. Connexin43 immunoreactivity appeared along the smooth muscle cell surface in the outer circular layer of the small intestine of both animals. Connexin 40 immunoreactivity was not observed in the muscle layer other than in the wall of large blood vessels. It is suggested that connexin45-expressing cells along the deep muscular plexus of dog and rat small intestine are likely to act as a constituent of a pacemaker system, which may include a conductive system, by forming a cellular network operating via specific types of gap junctions.

Changes in the phosphorylation states of connexin43 in myoepithelial cells of lactating rat mammary glands.

Using specific antibodies and cDNA probe, we examined the expression pattern of a major gap junction protein, connexin43 (Cx43), in rat mammary glands during pregnancy and lactation. Double immuno-fluorescence revealed that the labeling of Cx43 was superimposed in the alpha-smooth muscle actin-positive cells, suggesting that myoepithelial cell were interconnected by gap junctions formed of Cx43. Just after delivery, the Cx43-labeled plaques were enlarged and increased in intensity. Northern and Western blot analyses confirmed the dramatic induction of Cx43 at both mRNA and protein levels on the day of parturition. Cx43 mRNA transcript immediately declined, while the increase of Cx43 protein continued for a few days. During pregnancy, immunoblots showed two bands of almost equal amounts at 43 and 45 kDa. Following delivery, the 45-kDa band gradually increased in intensity with a concomitant decrease of the 43-kDa band. From the sixth day of lactation, Cx43 was always detected as a single band at 45 kDa. Alkaline phosphatase treatment of immunoprecipitated Cx43 revealed that both bands represented phosphorylated forms, thus indicating that Cx43 was naturally phosphorylated and that it altered its phosphorylation states during lactation stages. These results suggest that the induction of Cx43 with the changes in the phosphorylation states plays an important role in the lactating function of myoepithelial cells in rat mammary glands. This is the first report on the changes of Cx43 phosphorylation states during physiological stages in vivo.

Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation.

We examined the dynamics of connexins, E-cadherin and alpha-catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or alpha-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

A single point mutation of hamster aminoacyl-tRNA synthetase causes apoptosis by deprivation of cognate amino acid residue.

BACKGROUND: We have isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line derived from the golden hamster (Nishimoto & Basilico 1978; Nishimoto et al. 1982). Using these mutants as a recipient of DNA-mediated gene transfer, we have been cloning human genes which complement these ts mutants. RESULTS: Cultures of tsBN269 cells, a temperature-sensitive mutant of the BHK21 cell line, underwent apoptosis at 39.5 degrees C, a nonpermissive temperature. The gene complementing the tsBN269 cells was cloned and found to encode lysyl-tRNA synthetase. Indeed, tsBN269 cells were found to have a single cytosine to a thymine point mutation at the first nucleotide of codon 542 in hamster lysyl-tRNA synthetases. Due to this mutation, the activity of lysyl-tRNA synthetase was reduced--even at 33.5 degrees C, a permissive temperature. Consistent with these findings, while supplementation with lysine permitted tsBN269 cells to grow at a nonpermissive temperature, the deprivation of lysine caused apoptosis in tsBN269 cells, even at 33.5 degrees C. Cycloheximide inhibited the apoptosis caused by lysine starvation at 33.5 degrees C, but not at 39.5 degrees C. We also found that another hamster temperature-sensitive mutant, tsBN250, which is defective in histidyl-tRNA synthetase, entered apoptosis with the deprivation of histidine. CONCLUSION: Our data suggested that the defect in aminoacyl-tRNA synthetase turned on the cascade of apoptosis that was already present in the cells.

Connexin 43 and the glucose transporter, GLUT1, in the ciliary body of the rat.

To investigate the relationship between the gap junction protein connexin 43 and the glucose transporter GLUT1, their localization was visualized by double-immunofluorescence microscopy using frozen sections as well as immunogold staining of ultrathin frozen sections. In pigmented epithelial cells, most of the GLUT1 was localized along the plasma membrane facing the blood vessels, whereas in non-pigmented epithelial cells, it was present along the plasma membrane facing the aqueous humor. Connexin 43 was abundant in the ciliary body and localized mainly in the gap junctions connecting the pigmented and non-pigmented epithelial cells. Localization of GLUT1 and connexin 43 in the blood-aqueous barrier suggests that GLUT1, connexin 43, and GLUT1 disposed in this order could be a machinery responsible for the transport of glucose across the blood-aqueous barrier.