Application of a barometer for assessment of oral functions: Donders space.

We developed a barometer applicable to a small space, to assess oral and pharyngeal functions. Negative oral pressure during rest and pressure changes during swallowing were measured in a space between the palate and tongue (STP). Twenty volunteers were asked to sit in a chair in a relaxed upright position. A sensor was placed on the posterior midline of hard palate. Recording commenced just before subjects closed their lips and continued. Subjects were asked to swallow saliva and keep the apposition. Finally, subjects were asked to open their mouth. Recordings were performed five times, and 5 s of continuous data in each phase was averaged. To verify the reliability of the system, the same procedure was accomplished with twin sensors. When the jaw and lips were closed, the pressure slightly decreased from atmospheric pressure (-0·17 ± 0·24-kPa). After swallowing, the pressure in STP showed more negative value (-0·50 ± 0·59-kPa). There is a significant difference between the values in open condition and after swallowing (P < 0·001) and between values after swallowing and final open condition (P < 0·05). Twin sensor showed almost the same trajectories of pressure changes for all the recordings. Obtained negative pressure might generate about 0·71-N of force and would be enough to keep the tongue in the palatal fossa at rest. The system detected large negative/positive pressure changes during swallowing. We conclude this system may be a tool to evaluate oral functions.

Prolongation of somitogenesis in two anguilliform species, the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus, with refined descriptions of their early development.

The embryonic development of the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus was morphologically investigated with laboratory-reared specimens to clarify the characteristics of somitogenesis. In A. japonica, somites were first observed at 18 h post fertilization (hpf) when epiboly reached 90%. Somitogenesis progressed at a rate of 1·6 h-1 at mean ± s.d. 22·6 ± 0·7° C and completed at 107 hpf (3 days post hatching; dph) when total number of somites (ST) reached 114, which corresponds to the species' number of vertebrae (112-119). In M. cinereus, somites were first observed at 14 hpf when epiboly completed. Somitogenesis progressed at a rate of 1·9 h-1 at mean ± s.d. 24·4 ± 0·2° C and completed at 90 hpf (2 dph) with 149 ± 4 ST, which corresponds to the species' number of vertebrae (142-158). Both species hatched before somitogenesis was completed, at 37 hpf with 47 ST and 42 hpf with 82 ± 4 ST, respectively. The formation of other organs such as the heart, mouth and pectoral fin bud occurred during somitogenesis. Comparison with the development of zebrafish Danio rerio indicates a prolongation of somitogenesis in A. japonica and M. cinereus. Their somitogenesis rates, however, correspond well with that of D. rerio estimated at the same temperature and their developmental stages at hatching are almost equivalent to other fishes having similar yolk sizes. Therefore, the prolongation of somitogenesis in A. japonica and M. cinereus may be accounted for solely by the increased numbers of somites to be formed, not by a slow somitogenesis rate or an acceleration in organogenesis.

Effective three-dimensional evaluation analysis of upper airway form during oral appliance therapy in patients with obstructive sleep apnoea.

The oral appliance (OA) is considered to be an effective treatment modality for obstructive sleep apnoea (OSA). Upper airway enlargement during OA therapy is critical, and lateral cephalometry has been used for the two-dimensional evaluation of upper airway form during this therapy. However, this method cannot provide an accurate three-dimensional (3D) view of upper airway form. To confirm the effects of OA on the upper airway in patients with OSAS, we performed CT in the presence and absence of OA in 15 Japanese patients (12 males, 3 females) who responded to OA therapy. CT in the presence and absence of OA was consecutively performed for each patient, and upper airway cross-sectional area in six arbitrary planes parallel to the palatal plane was measured. Next, 3D image reconstruction was performed; morphological changes in upper airway form were evaluated, and upper airway volume at three levels from the palatal plane to the deepest point of the epiglottis was measured. The cross-sectional area of two planes in the posterior soft palate region significantly increased in the presence of OA compared with that in the absence of OA. In the presence of OA, upper airway cross-sectional area and volume significantly increased in the posterior soft palate region compared with those in the posterior tongue region. 3D CT image reconstruction accurately confirmed morphological changes in the upper airway during OA therapy. Continued use of this 3D evaluation is expected to improve the results of OA therapy in the future.

Estimation of the absorption coefficients of two-layered media by a simple method using spatially and time-resolved reflectances.

Our newly developed method using spatially and time-resolved reflectances can easily estimate the absorption coefficients of each layer in a two-layered medium if the thickness of the upper layer and the reduced scattering coefficients of the two layers are known a priori. We experimentally validated this method using phantoms and examined its possibility of estimating the absorption coefficients of the tissues in human heads. In the case of a homogeneous plastic phantom (polyacetal block), the absorption coefficient estimated by our method agreed well with that obtained by a conventional method. Also, in the case of two-layered phantoms, our method successfully estimated the absorption coefficients of the two layers. Furthermore, the absorption coefficients of the extracerebral and cerebral tissue inside human foreheads were estimated under the assumption that the human heads were two-layered media. It was found that the absorption coefficients of the cerebral tissues were larger than those of the extracerebral tissues.

Identification of the Novel Tooth-Specific Transcription Factor AmeloD.

Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs' development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like ( ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell-cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

In vitro bioactivity evaluation of titanium and niobium metals with different surface morphologies.

Current orthopaedic biomaterials research mainly focuses on designing implants that could induce controlled, guided and rapid healing. In the present study, the surface morphologies of titanium (Ti) and niobium (Nb) metals were tailored to form nanoporous, nanoplate and nanofibre-like structures through adjustment of the temperature in the alkali-heat treatment. The in vitro bioactivity of these structures was then evaluated by soaking the treated samples in simulated body fluid (SBF). It was found that the morphology of the modified surface significantly influenced the apatite-inducing ability. The Ti surface with a nanofibre-like structure showed better apatite-inducing ability than the nanoporous or nanoplate surface structures. A thick dense apatite layer formed on the Ti surface with nanofibre-like structure after 1 week of soaking in SBF. It is expected that the nanofibre-like surface could achieve good apatite formation in vivo and subsequently enhance osteoblast cell adhesion and bone formation.

Oral behavior from food intake until terminal swallow.

We analyzed oral behavior from food intake until terminal swallow for mastication and swallowing under a freely eating condition with a natural food. Measurements, including movement of the mandible and tongue, the size of the gape, different sequences involved in the oral aspect of the swallowing action, and bolus size and movement were carried out in five "freely eating subjects" using videofluorography. During food intake, the tongue moved forwards and backwards to introduce food into the mouth, to compress the food against the hard palate, and to transport food to the occlusal surface of the molar teeth. Most of the food was swallowed in the first swallow, and any residual food was aggregated by the tongue into a bolus and then swallowed in the last swallow. These findings suggest that 1) tongue manipulation plays an important role in recognizing and evaluating the volume of bite taken, 2) the intra-oral compression of food has a role in the recognition of food texture, 3) stage I transport is closely bound to the texture recognition process, 4) humans need at least two swallows, even with one bite of food, when ingesting food freely, and 5) the duration time of the oral stage of swallowing may depend on the bolus volume and be longer for smaller volumes unlike those measured under the command swallow.

Alveolar cleft osteoplasty using tissue-engineered osteogenic material.

The use of tissue-engineered osteogenic material comprising platelet-rich plasma and autologous mesenchymal stem cells isolated, expanded and induced to osteogenic potential in bone augmentation procedures as a replacement for autologous bone grafts, offers predictable results with minimal donor-site morbidity. This material was applied for an alveolar cleft osteoplasty of a 9-year-old female patient. Serial computed tomograms showed the regenerated bone extending from the cleft walls after 3 months and bridging the cleft after 6 months, with 79.1% of the grafted region after 9 months at the time when the canine and lateral incisor in the affected side erupted in the reconstructed alveolar ridge.

The mode of actions of lysozyme as an immunoglobulin production stimulating factor.

As we demonstrated before, hen egg white lysozyme stimulates immunoglobulin production by a human-human hybridoma line, HB4C5 cells and human peripheral blood lymphocytes. Then, the mode of actions of lysozyme as an immunoglobulin production stimulating factor was investigated. The immunoglobulin production stimulating activity of lysozyme was inactivated by trypsin digestion, even though the enzymatic activity was completely preserved. This fact suggests that the immunoglobulin production stimulating effect of lysozyme is irrelevant to its enzymatic function. Furthermore, this means that the effect is a novel function of this enzyme. Lysozyme enhanced IgM production by transcription-suppressed HB4C5 cells treated with actinomycin D. However, the enzyme was ineffective to accelerate IgM production by translation-suppressed HB4C5 cells treated with cycloheximide or sodium fluoride. In addition, the intracellular IgM content of HB4C5 cells treated with monensin for suppression of the post-transcription activity was obviously increased by lysozyme, although the secretion of IgM was inhibited. These findings suggest that lysozyme accelerates the translation process to enhance immunoglobulin productivity.

Ameloblastin expression in rat incisors and human tooth germs.

We recently identified ameloblastin as an ameloblast-specific gene product from a rat incisor cDNA library (Krebsbach et al., J. Biol. Chem. 271: 4431-4435, 1996). Here we report the developmental pattern of expression of ameloblastin in rat incisors and human tooth germs as visualized by in situ hybridization and immunochemistry. Compared to the expression of amelogenin, the major ameloblast product, ameloblastin mRNA was more widely expressed in ameloblasts from the presecretory to the late maturation stage of development. Ameloblastin mRNA was first observed in the juxtanuclear cytoplasm or presecretory stage ameloblasts, gradually increased in the distal cytoplasm of secretory stage ameloblasts and was found throughout the cytoplasm of early to late maturation stage ameloblasts. The immunostaining of ameloblastin, using a monospecific antibody raised against a recombinant protein, showed intense reactivity in Tomes' processes of secretory stage ameloblasts and surrounding enamel. The immunoreaction was concentrated in the juxtanuclear cytoplasm of late maturation stage ameloblasts. High-resolution colloidal gold immunocytochemistry established the presence of ameloblastin antigenicity in the Golgi apparatus, secretory granules in Tomes' process and enamel. Human tooth germs in early to late bell stage also expressed ameloblastin mRNA and ameloblastin antigenicity in the ameloblasts. Western blot analysis of protein extracts from rat incisor tissues indicated that ameloblastin can be found in the enamel epithelial tissue and in mineralized enamel, as well as in the EDTA decalcification solution. These data indicate that ameloblastin is an ameloblast secretory product which is sequentially expressed from the presecretory to the late maturation stage in rat and human teeth. This unique developmental pattern suggests that ameloblastin may have a broader role in amelogenesis than amelogenin and tuftelin.

Biological activities and quantitative structure-activity relationships of spiro[imidazolidine-4,4'(1'H)-quinazoline]-2,2',5(3'H)-triones as aldose reductase inhibitors.

A series of spiro[imidazolidine-4,4'(1'H)-quinazoline]- 2,2'5(3'H)-triones were prepared and tested for aldose reductase inhibitory activity. The 6'-halogenated derivatives were found to be highly potent in vitro inhibitors of male rabbit lens aldose reductase and in vivo inhibitors of polyol accumulation in the sciatic nerves of galactosemic rats. Of these, (4R)-6'-chloro-3'-methylspiro[imidazolidine-4,4'(1'H)-quinazoline] -2,2',5(3'H)-trione (67) showed the most potent in vitro and in vivo activities. An oral dose of 3 g/kg of compound 67 caused neither death nor behavioral abnormality in the preliminary acute toxicity study using mice and rats. Compound 67 was selected as a candidate for further evaluation. The quantitative structure-activity relationships in this series are also discussed.

Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors.

Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and mid- to late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 microm to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. (J Histochem Cytochem 46:911-934, 1998)

De novo balanced translocation (6;18)(q21;q21.3 or q22) [corrected] in a patient with heterotaxia.

We report on a sporadic case of heterotaxia with a de novo chromosome structural abnormality. The patient had inversely located heart (dextrocardia), stomach, duodenum, and cecum. In addition, she had cerebral atrophy, hypertelorism with telecanthus, infraorbital skin furrows, ear-lobe grooves, prominent maxilla and teeth, large carp mouth, short fifth fingers with limited flexion, generalized hypotonicity, and severe psychomotor retardation. High-resolution chromosome banding analysis demonstrated an apparently balanced translocation: 46,XX,t(6;18)(q21;q21.3). It is hypothesized that both heterotaxia and the chromosomal abnormality in the patient are causally related and a putative situs determining gene has been disrupted by the chromosome break, i.e., a position effect or a cryptic deletion at around the breakpoints. The translocation in our patient may be a good source for positional cloning of the gene.

Amplitudes of accommodation of primate lenses refilled with two types of inflatable endocapsular balloons.

OBJECTIVE: To investigate the maintenance of ocular accommodation by refilling the lenses of the eyes of youthful primates with inflatable endocapsular balloons. METHODS: The lenses of 21 cynomolgus monkeys were refilled following endocapsular phacoemulsification with a balloon that either approximates the shape of the non-accommodated lens or the accommodated crystalline lens. RESULTS: In nine of 15 successfully refilled lenses, we were able to perform automated refractometry to determine the amplitude of accommodation, ie, changes in refraction before and 1 hour after application of topical 4% pilocarpine hydrochloride to alter lens shape. At 2 weeks, 2 to 3 months, and 6 to 12 months after operation, mean (+/- SD) accommodation was 4.6 +/- 2.5, 2.5 +/- 0.5, and 1.7 +/- 0.7 diopters (D), respectively, in the lenses refilled with the nonaccommodation balloon (n = 5; preoperative value, 15.2 +/- 1.3 D), and it was 1.9 +/- 0.5, 1.3 +/- 0.9, and 1.8 +/- 0.9 D, respectively, in the lenses refilled with the accommodation balloon (n = 4; preoperative value, 17.0 +/- 2.9 D). CONCLUSION: The greater yield of accommodation with the nonaccommodation balloon is consistent with the recent theory on the mechanism of accommodation. Although the obtained accommodation was a small fraction of values determined prior to the operation and the small amplitude of accommodation decreased over time, the feasibility of refilling the lens with an inflatable endocapsular balloon, allowing at least some accommodation in the eyes of youthful primates, was demonstrated. Applied to humans, this procedure may allow accommodation following cataract surgery.

Efficacy of equator rings in an experimental rabbit study.

OBJECTIVE: To determine the efficacy of the equator ring in maintaining the circular contour of the capsule bag equator and transparency of the posterior capsule after crystalline lens extraction in 12 rabbit eyes. METHODS: After phacoemulsification through a 5-mm window made by continuous circular capsulorrhexis in the central anterior capsule, a flexible silicone ring (outer diameter, 11.0 mm; width, 1.5 mm; and thickness, 1.5 mm) was implanted in the capsule in seven rabbit eyes. A 13.0-mm Sinskey-style posterior chamber intraocular lens was then implanted inside the ring. Three other eyes received only equator rings, and two others received only intraocular lenses. Eyes were followed up for 3.5 +/- 1.4 months (mean +/- SD) before enucleation and evaluation by stereomicroscopy; they then underwent light microscopic examination. RESULTS: The two eyes that received only the intraocular lens had equatorial distortion and severe posterior capsule opacification. In nine (90%) of the 10 eyes that were implanted with the rings, the circular contour of the equator was preserved, and seven (70%) of these 10 eyes had transparent posterior capsules. CONCLUSION: Implantation of the equator ring effectively maintained the circular contour of the capsule bag equator and inhibited cells at the bag equator from proliferating toward the center of the posterior capsule in the rabbit.

Biological response of chondrocytes to hydrogels.

Primary bovine chondrocytes were encapsulated in alginate and alginate combined with cartilage matrix extract, Cartrigel, for the purpose of cartilage tissue engineering. The cell constructs were incubated in vitro and gene expression of cartilage-specific extracellular matrix molecules was quantitated and localized with in situ hybridization with a decrease in expression observed in the alginate-Cartrigel constructs. Further understanding of cell response to scaffolds will allow rational design and development of hydrogels for cartilage tissue engineering.

Regional mapping of gas transport during high-frequency and conventional ventilation.

The effects of changing tidal volume (VT) and frequency (f) on the distribution of ventilation during high-frequency ventilation (HFV) were assessed from the washout of nitrogen-13 by positron emission tomography. Six dogs, anesthetized and paralyzed, were studied in the supine position during conventional ventilation (CV) and during HFV at f of 3, 6, and 9 Hz. In CV and HFV at 6 Hz, VT was selected to achieve eucapnic arterial partial pressure of CO2 (37 +/- 3 Torr). At 3 and 9 Hz, VT was proportionally changed so that the product of VT and f remained constant and equal to that at 6 Hz. Mean residence time (MRT) of nitrogen-13 during washout was calculated for apical, midheart, and basal transverse sections of the lung and further analyzed for gravity-dependent, cephalocaudal and radial gradients. An index of local alveolar ventilation per unit of lung volume, or specific ventilation (spV), was calculated as the reciprocal of MRT. During CV vertical gradients of regional spV were seen in all sections with ventral (nondependent) regions less ventilated than dorsal (dependent) regions. Regional nonuniformity in gas transport was greatest for HFV at 3 and 6 Hz and lowest at 9 Hz and during CV. During HFV, a central region at the base of the lungs was preferentially ventilated, resulting in a regional time-averaged tracer concentration equivalent to that of the main bronchi. Because the main bronchi were certainly receiving fresh gas, the presence of this preferentially ventilated area, whose ventilation increased with VT, strongly supports the hypothesis that direct convection of fresh gas is an important mechanism of gas transport during eucapnic HFV. Aside from the local effect of increasing overall lung ventilation, this central area probably served as an intermediate shuttle station for the transport of gas between mouth and deeper alveoli when VT was less than the anatomic dead space.

Changes in reflex responses of the masseter and digastric muscles during sleep in freely behaving rabbits.

The aim of this study was to clarify the modulation of motoneuron excitability in masticatory muscles during sleep. For this purpose, changes in the reflex responses of the masseter and digastric muscles between sleep and wakefulness were studied in freely behaving rabbits. Stimulation of the jaw closing muscle spindle afferents induced the masseteric monosynaptic reflex (MMR). During quiet sleep (QS), which gradually replaced quiet wakefulness, the mean MMR amplitude showed no change. During active sleep (AS). MMR amplitudes were markedly reduced, but they were confounded by the occasional occurrence of facilitation in the amplitude. The facilitatory reflexes were often related to rapid eye movements (REMs). However, the excitatory input probably did not originate from the same region as the REM generator, since the REM and the large MMR did not always occur simultaneously. On the other hand, jaw opening reflexes remained inhibited. The results indicated that there is mainly a difference in the excitability between the two groups of motoneurons during AS; masseter motoneuron activity was inhibited but occasionally facilitated by excitatory inputs occurring in association with REMs, however, the digastric motoneuron activity was remained inhibited. The excitatory inputs may induce dysfunctional muscle contraction of the jaw closing muscles as seen in bruxism.

Changes in jaw reflexes by stimulation of the hypothalamus in anesthetized rabbits.

Changes in the masseteric monosynaptic reflex (MMR) and jaw-opening reflex (JOR) responses resulting from conditioning stimulation in the hypothalamus were studied in anesthetized rabbits. Stimulation of the lateral hypothalamus evoked a facilitation of the MMR and an inhibitory or facilitatory effect on the JOR. The facilitatory effect on JOR was stronger than that on the MMR. The facilitatory effective site for the JOR was in the dorsal and lateral directions as compared to the inhibitory field. The results suggest two functionally distinct regions in the lateral hypothalamus that separately project to the jaw-opening muscles.

Two-dimensional jaw tracking and EMG recording system implanted in the freely moving rabbit.

A system for simultaneously recording mandibular position in the sagittal plane together with masticatory muscle activity was designed and tested in rabbits. Two small magnetic sensors were implanted in the maxillary bone and a powerful magnet made of a rare earth metal attached to the mandibular central incisors. The magnetic sensors detected the mandibular movements in the sagittal plane by movement of the magnet. Masseter EMG was recorded by fine wire electrodes and amplified by a specially designed amplifier. The necessary preamplifiers were assembled as an integrated circuit (IC) chip in a small housing. The signals from the preamplifier were then passed through a signal processing unit and taped on an instrumentation tape. The system was applied to the freely moving rabbit supplied with food and water during the night. It worked without any trouble for more than 24 h. Since the implanted magnetic sensors were stable for more than 4 months, long-term recording could be done by merely reimplanting the magnet, the cables and the EMG electrodes, which was simple.