Seiichiro Kitamura

AUTOFLUORESCENCE IN HUMAN DENTINE IN RELATION TO AGE, TOOTH TYPE AND TEMPERATURE MEASURED BY NANOSECOND TIME-RESOLVED FLUORESCENCE MICROSCOPY

Stationary and dynamic characteristics of autofluorescence in human dentine were examined with a nanosecond time-resolved fluorescence microphotometer. With ageing, the intensity of the fluorescence increased whereas the decay time decreased. There were no differences in fluorescence intensity, decay time or spectral profile among tooth types taken from the same individuals or from individuals of the same age. Fluorescence intensity was enhanced after the application of heat (37 and 50 degrees C), but remained unchanged when specimens were kept at 4 degrees C. These results suggest that the increase in fluorescence intensity was partially due to a temperature-dependent physicochemical mechanism.

Location of cell somata and the peripheral course of axons of the geniohyoid and thyrohyoid motoneurons: A horseradish peroxidase study in the rat

The location of cell somata of the motoneurons supplying the rat geniohyoid and thyrohyoid muscles and the peripheral course of their axons were studied by intramuscular injection of horseradish peroxidase in combination with severing the innervating nerves of these muscles. Labeled geniohyoid motoneurons were found ipsilaterally in the ventrolateral subnucleus of the hypoglossal nucleus, which separates at midlevel of this nucleus from the main hypoglossal nucleus lying near the central canal and finally projects to the dorsomedial part of the ventral horn in the transition level of the brain/spinal cord. Geniohyoid motoneuron axons mostly pass through the hypoglossal nerve, whereas axons of the motoneurons in the transition level, though much fewer in number, pass through the first cervical nerve. Labeled thyrohyoid motoneurons were found ipsilaterally to form a cell column that extends from the caudal end of the main hypoglossal nucleus in the transition level of the brain/spinal cord to the dorsomedial part of the ventral horn in the caudal first cervical segment. All their axons passed through the first cervical nerve, and thus, the rostral portion of the thyrohyoid motoneuron column, which appears to be part of the hypoglossal nucleus, is not included in this nucleus but constitutes the supraspinal nucleus, which connects the hypoglossal nucleus to the ventral horn motoneuron group. The mean cell body sizes of the geniohyoid and thyrohyoid motoneurons were 28.7 +/- 4.4 and 29.3 +/- 4.6 micrometers, respectively, and their cell body size distributions were unimodal.

The Central Projections of the Monkey Tooth Pulp Afferent Neurons

Transganglionic transport of horseradish peroxidase conjugated to wheatgerm agglutinin (HRP:WGA) entrapped in hypoallergenic polyacrylamide gel was used to study the patterns of termination of primary afferents that innervate the upper and lower tooth pulps within the trigeminal sensory nuclear complex (TSNC) of the monkey. HRP:WGA injections were also made into the lower incisors and molars, in order to examine the topographic arrangement of pulpal afferent projections. HRP-labeled pulpal afferents innervating lower and upper teeth projected ipsilaterally to the rostral subnucleus dorsalis (Vpd) and caudal subnucleus ventralis (Vpv) of the nucleus principalis (Vp); the rostrodorsomedial (Vo.r) and dorsomedial (Vo.dm) subdivisions of the nucleus oralis (Vo); the dorsomedial subdivision of the nucleus interpolaris (Vi); and laminae I-II and/or V of the nucleus caudalis (Vc) at its rostralmost level. The HRP-labeled terminals from upper and lower pulpal afferents formed a rostrocaudal column from the midlevel of Vp to the rostral tip of Vc. The label in Vp and Vo was considerably dense, but the column of terminals was interrupted at the Vpd-Vpv transition. The label in Vi and Vc was much less dense compared to that in the rostral nuclei, and the column of terminals was interrupted frequently. The representation of the upper and lower teeth in TSNC was organized in a somatotopic fashion that varied from one subdivision to the next, though their terminal zones overlapped within Vpd. The upper and lower teeth were represented in Vpv, Vo.r, Vo.dm, Vi, and Vc in a ventrodorsal, dorsoventral, lateromedial, lateromedial, and lateromedial sequence, respectively. Topographic arrangement was also noticed for the projections of pulpal afferents from the lower incisors and molars: The representations of the lower incisors and molars in Vpv, Vo.r, Vo.dm, Vi, and Vc were organized in a lateromedial, dorsoventral, ventrodorsal, ventrodorsal, and lateromedial sequence, respectively. The present results indicating sparse projections from pulpal afferents in the monkeys Vc are in good correspondence with a clinical report that trigeminal tractotomy just rostral to the obex has no significant effect on dental pain perception in patients. Furthermore, the present study indicates that projection patterns of pulpal afferents--which include the termination sites, the density of terminations between nuclei, and topographic arrangement--differ among animal species.

Cationic uncouplers of oxidative phosphorylation are inducers of mitochondrial permeability transition

To determine whether cationic uncouplers of oxidative phosphorylation induce permeability transition in mitochondria, the effects of the divalent cationic sulfhydryl cross‐linker copper‐o‐phenanthroline (Cu(OP)2) and the cyanine dye tri‐S‐C4(5) on rat liver mitochondria were examined. Like Ca2+, they accelerated mitochondrial respiration with succinate and induced mitochondrial swelling when inorganic phosphate (Pi) was present in the incubation medium. The acceleration of respiration and swelling were inhibited by the SH‐reagent N‐ethylmaleimide, and by the specific permeability transition inhibitor cyclosporin A (CsA). In addition, these cations, like Ca2+, induced release of ADP entrapped in the mitochondrial matrix space, and the morphological change of mitochondria induced by these cations was essentially the same as that induced by Ca2+. It is concluded that the uncoupling actions of Cu(OP)2 and tri‐S‐C4(5) are due to induction of permeability transition in the inner mitochondrial membrane.

Variations in the tensor veli palatini muscle with special reference to its origin and insertion.

Objective Previous research on the tensor veli palatini muscle (TVP) has produced conflicting descriptions of its functions and topographical relationships with other orofacial structures. The goal of this study was to describe the morphology of the TVP in a systematic and comprehensive manner. Methods One hundred nineteen sides of 77 human heads from donated cadavers were partially dissected under a binocular microscope. Histological examination of the hard tissue–muscle interfaces was also undertaken. Results There were two adjacent origins of the TVP: the cranial base origin (CB origin) and the auditory tube cartilage origin (AT origin). The CB origin always lay anterior to the AT origin and there was no septum or loose tissue between the two muscular laminae leading from these origins. The muscle fibers converged on a central tendinous plate in the muscle belly, which gradually became a common tendon that rounded the pterygoid hamulus before inserting into the palatine aponeurosis. Notably, secondary insertions were found on the maxillary tuber (33.6%) and/or in the submucosal tissue near the palatoglossal arch (37.8%). Maxillary insertions were almost exclusively associated with an AT origin that was wide as or wider than the CB origin. Histological observations confirmed that the hamulus acted purely as a pulley and suggested that a connecting band to the tensor tympani had no or few functions of an intermediate tendon. Conclusions The TVP appears to act as the dilator tubae and that this function can be maintained by preserving or reconstructing the maxillary insertion during push-back surgery, even if hamulotomy is necessary.

Localization of rabbit laryngeal motoneurons in the nucleus ambiguus

The localization of rabbit laryngeal motoneurons in nucleus ambiguus was investigated using injection of a fluorescent labeling substance, i.e., nuclear yellow, into the individual laryngeal muscles. The nucleus ambiguus of the rabbit comprises four subnuclei, CoG, SGm, SGl, and DiG. The CoG is a group of compactly arranged neurons, and is situated in the rostral one-half of the nucleus. The SG, situated in its rostral one-third, is scattered around the CoG, with a subdivision into SGm and SGl. These subdivisions are medial and lateral to the CoG, respectively. The DiG is formed by diffusely arranged neurons, and is located in the caudal two-thirds of the nucleus. All labeled motoneurons were found in the ipsilateral nucleus ambiguus. The motoneurons supplying the cricothyroid muscle, which is innervated by the superior laryngeal nerve, were present in the SGm, with a clear rostralward segregation from the other motoneurons. The motoneurons supplying the muscles innervated by the inferior laryngeal nerve were located in the DiG, where they displayed a rostrocaudal myotopical arrangement in the order posterior cricoarytenoid, thyroarytenoid, and lateral cricoarytenoid. The posterior cricoarytenoid motoneurons were intermingled with the thyroarytenoid motoneurons in the rostral two-thirds of the DiG, and the former tended to be concentrated more rostrally than the latter. The lateral cricoarytenoid motoneurons were confined to the most caudal one-third of the DiG.

Influenza vaccine with Surfacten, a modified pulmonary surfactant, induces systemic and mucosal immune responses without side effects in minipigs.

Immune responses and side effects of intranasally administered flu vaccine with the commercial product Surfacten, a modified bovine pulmonary surfactant, were investigated in minipigs. The use of minipigs was based on the anatomical resemblance of nasal lymph nodes, the principal antigen uptake site of respiratory mucosal immunity, between pig and human. Intranasal instillation of HA vaccine adjuvanted with Surfacten elicited significantly higher serum hemagglutination inhibition titers than the antigen alone, with wide cross-neutralizing activities of secretory IgA in nasal washes. No significant induction of inflammatory cytokines or migration of inflammatory cells was observed at the site of immunization or serum after the first immunization. These data suggest the potential usefulness of Surfacten for mucosal vaccination.

Okadaic acid induces tyrosine phosphorylation of IκBα that mediated by PKR pathway in human osteoblastic MG63 cells

Treatment of human osteosarcoma cell line MG63 cells with okadaic acid stimulated phosphorylation of IκBα, as judged from the results of Western blot analysis and a λ protein phosphatase dephosphorylation assay. The stimulated phosphorylation of IκBα was both time- and dose-dependent. The phosphorylation sites of IκBα were taken to be tyrosine residues because the anti-phospho-tyrosine antibody bound to the samples immunoprecipitated with the anti-IκBα antibody. In the cells treated with 100 nM okadaic acid consequential translocation of NF-κB p65 from the cytosol to the nucleus occurred. Double-stranded RNA-dependent protein kinase (PKR) is a player in the cellular antiviral response and is involved in transcriptional stimulation through activation of NF-κB. We investigated the functional relationship between PKR and IκBα phosphorylation by constructing MG63 PKR K/R cells that produced a catalytically inactive mutant PKR. NF-κB p65 was detected in the nucleus of these cells, even in the unstimulated cells. Although IκBα was degraded phosphorylation of eIF-2α, a substrate of PKR, did not occur in the mutant cells treated with okadaic acid. Our results suggest that okadaic acid-induced tyrosine phosphorylation of IκBα was mediated by PKR kinase activity, thus indicating the involvement of this kinase in the control mechanism governing the activation of NF-κB. (Mol Cell Biochem 276: 211–217, 2005)

A horseradish peroxidase study of rat lingual motoneurons with axons passing through the cervical nerve

The peripheral course of axons of rat lingual motoneurons was studied by HRP injection into the hypoglossal nerve in combination with transecting of the hypoglossal and/or cervical nerve components of the hypoglossocervical plexus. Furthermore, soma sizes of labeled lingual motoneurons were compared in transverse section with those of labeled geniohyoid and thyrohyoid motoneurons, which are situated adjacent to the lingual motoneurons. We found that axons of the majority of lingual motoneurons lying in the main hypoglossal nucleus passed through the hypoglossal nerve throughout their course to the tongue. In a remaining small number of lingual motoneurons lying in a medial portion of the ventromedial subnucleus in the caudal fourth of the main hypoglossal nucleus, their axons passed through the first cervical nerve to the upper root of the ansa cervicalis to the hypoglossal nerve and then to its medial branch. The labeled lingual motoneurons with axons passing through the cervical nerve were intermingled with those whose axons passed through the hypoglossal nerve. The latter motoneurons, however, diminished in number while being traced caudally, and finally in the most caudal main hypoglossal nucleus the former motoneurons occupied a major part of this nucleus. The lingual motoneurons with axons passing through the cervical nerve were smaller in soma size than those with axons passing through the hypoglossal nerve. These two types of lingual motoneurons were both smaller in soma size than the geniohyoid and thyrohyoid motoneurons, and their soma shape was not as flat as that of the latter types of motoneurons.

Mastoparan peptide causes mitochondrial permeability transition not by interacting with specific membrane proteins but by interacting with the phospholipid phase

The mastoparan peptide is known as an inducer of the mitochondrial permeability transition. Although mastoparan was suggested to interact with a proteinaceous target in mitochondria to induce this transition, the action sites of mastoparan have not yet been investigated. To clarify whether specific interactions of mastoparan with receptors or enzymes are associated with the induction of this permeability transition, we examined the effects of d‐isomeric peptides, which were synthesized using d‐amino acids assembled in endogenous (inverso mastoparan) and reverse (retro‐inverso mastoparan) orientations. When we added inverso mastoparan to isolated mitochondria, the peptide caused the permeability transition in a partially cyclosporin A‐sensitive manner at lower doses and in a cyclosporin A‐insensitive manner at higher ones. The manners of action and the potencies of inverso mastoparan were close to those of parent mastoparan, indicating that the targets of mastoparan for induction of the permeability transition were neither receptors, nor enzymes in the mitochondria. Retro‐inverso mastoparan also had the same effect on the mitochondria as mastoparan, although the potencies of the effect were weaker. Not only on mitochondria, but also on phospholipid vesicles, mastoparan and inverso mastoparan showed massive permeabilization effects at the same potencies, although retro‐inverso mastoparan showed weaker ones. These results indicate that mastoparan interacted with the phospholipid phase of the mitochondrial membrane (and not with specific proteins) to induce the permeabilization in cyclosporin A‐sensitive and ‐insensitive manners.