Endosomes along the degradation pathway exhibit a characteristic multivesicular organization, resulting from the accumulation of small vesicles or other membranous structures within the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated within lumenal vesicles through the action of Hrs and ESCRT complexes, a process that contributes to terminate signaling. Then, vesicles and their protein cargo are transported towards lysosomes for degradation. However, evidence also shows that lumenal vesicles can undergo “back-fusion” with the late endosome limiting membrane, a route presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. Vesicular stomatitis virus and anthrax toxin exploit this lumenal transport pathway to reach the cytoplasm. The back-fusion process depends on the late endosomal lipid lysobisphosphatidic acid or bis (monoacylglycero) phosphate and its putative effector Alix/AIP1. In this review, we will discuss intra-endosomal transport routes in mammalian cells, and in particular the mechanisms that are presumably involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

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The investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins (1,4,5)P₃] analogs has led to the isolation of a novel protein with a molecular size of 130 kDa, characterized as a molecule with a domain organization similar to phospholipase C (PLC)-δ1 but lacking enzymatic activity. Two isoforms of the molecule were subsequently identified, the molecule has been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins (1,4,5)-P₃ via the pleckstrin homology domain, the involvement of PRIP-1 in Ins (1,4,5)P₃-mediated Ca²⁺ signaling was first examined. Yeast two-hybrid screening of a brain cDNA library identified GABARAP (GABA_A receptor-associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible neurological involvement of PRIP, particularly in GABA_A receptor signaling. PRIP-1 and -2 double knock-out (DKO) mice were analyzed for GABA_A receptor function with special reference to the action of benzodiazepines whose target is the γ subunit of the receptors; sensitivity to benzodiazepine was reduced, as assessed by biochemical, electrophysiological, and behavioral analyses of DKO mice, suggesting the dysfunction of γ2 subunit-containing GABA_A receptors. The mesencephalic trigeminal nucleus, which mediates perceptions from periodontal mechanoreceptors and jaw-closer muscle spindles, receives many synaptic inputs, including those from GABA_A receptors, indicating that PRIP might indirectly be involved in rhythmical jaw movement. In the present article, we summarize our current research and the functional significance of PRIP.

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Dopamine (DA) is a major central nervous system (CNS) neurotransmitter with many important physiological activities. Investigations into the neuroanatomy and neurologic functions of the dopaminergic neural systems have generated much debate. Regarding neuroanatomy, physiological and pharmacological criteria have divided DA receptors into D1 and D2 subtypes. The genes encoding these subtypes have been cloned and classified into a D1 subfamily encompassing D1 and D5 receptors and a D2 subfamily with D2, D3, and D4. Based on the sequences of the cloned receptors, we prepared antibodies and riboprobes to elucidate the expression of the corresponding proteins and mRNAs in the rat area postrema (AP) by immunohistochemistry and in situ hybridization (ISH). The AP was obtained from adult male Sprague-Dawley rats undergoing brain surgery, and tissue samples were used for RT-PCR, immunohistochemistry, and ISH. The results showed that D2 and D5 receptors and their mRNAs exist in the rat AP. On the other hand, D1, D3, and D4 receptors and their mRNAs were not detected.

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Influences of tongue protrusion on rhythmical jaw movement were examined in lightly anesthetized adult rats. Both medial and lateral branches of the hypoglossal (XII) nerve on the right side were sectioned. The lateral branch of the XII nerve on the left side was also sectioned, and the medial branch was placed on stimulating electrodes. Intermittent electrical stimulation was delivered to the medial branch of the XII nerve to protrude the tongue, which was monitored by a tension-measuring device. Rhythmical jaw movements were elicited by electrical stimulation of the masticatory area of the cerebral cortex and were monitored using a magnet and magnet sensors. The following four major results were obtained: 1) the minimum opening position during cortically evoked chewing was significantly lowered by tongue protrusion, while no change was shown in the maximum opening position, 2) the lowering of the minimum opening position by tongue protrusion was consistent before and after mucosal anesthesia of the oral cavity, 3) the minimum and maximum opening positions were not altered by stimulation of the peripheral cut end of the medial branch of the XII nerve, and 4) the minimum opening position was lowered by stimulation of the central cut end of the medial branch of the XII nerve. These results suggest that the lowering of the minimum opening position by tongue protrusion is probably elicited from afferent signals through the medial branch of the XII nerve.

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The activities of Se-dependent glutathione peroxidases and non-Se-dependent glutathione peroxidase in the submandibular gland were observed using specific substrates. The activities for H₂O₂, cumene hydroperoxide, tert-butyl hydroperoxide, and phosphatidylcholine hydroperoxide were strongly inhibited by iodoacetate. After correction for the activity toward cumene hydroperoxide, it was shown that cumene hydroperoxide is mainly reduced by cytosolic glutathione peroxidase. Although the specific activity was lower than that of cytosolic glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase showed activity toward not only phosphatidylcholine hydroperoxide but also phosphatidylethanolamine hydroperoxide. These results suggest that cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase share a role in the reduction of hydroperoxide, but non-Se-dependent glutathione peroxidase (glutathione S-transferase) plays a lesser role.

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To assess the ability of the oral mucosa (tongue, palate, and buccal mucosa) to detect objects of different sizes, the accuracy of solid object size perception was measured in 14 healthy subjects. Five different sizes of steel spheres were used as objects for oral perception. First, the subjects were instructed to assess the size of the sphere, using the tip of the tongue, anterior hard palate, and one cheek without any visual or tactile information about the size of the testing sphere, and match the perceived size with a visual reference set of spheres placed in front of them (control experiment). The same procedure was repeated after local anesthesia (LA) of the tongue (T), tongue and palate (TP), and tongue palate and cheek (TPC), respectively. Two-point discrimination was tested on the tongue before and after LA to check its effect. Results showed a significant difference between two-point discrimination tests carried out with and without LA (paired t-test, p<0.05). One way repeated measures of ANOVA showed a significant (p<0.05) underestimation of the smallest sphere size in all groups except the control, and overestimation of a large sphere size with LA of the buccal mucosa. It was concluded that perception of the smallest sphere size could be affected by LA of the tongue alone, LA of both the tongue and palate, and LA of the tongue, palate, and cheek. Perception of the large sphere size (7.1 mm) was not affected by LA of the tongue alone or LA of both the tongue and palate. However, perception of this large sphere size (7.1 mm) was affected in the TPC experiment, in which cheek sensations were also lost.

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It is considered that osteoarthritis mainly affects articular cartilage, and the pathological mechanism involves both degenerative changes of the cartilage and bony changes. However, it has been indicated that the human temporomandibular joint has a higher capacity for remodeling. The causes and mechanisms of bony changes of human mandibular condyles have been a matter of speculation. In this study, we investigated human mandibular condyles by employing various microscopic investigations involving an electron probe micro analyzer and transmission electron microscope in addition to light microscopic observation. The results indicate that adaptive bony changes in the functional articular surface of mandibular condyles are brought about by cartilage calcification. Furthermore, the findings of cartilage calcification suggest the possibility of matrix-vesicle-mediated mineralization in the remodeling of aged human mandibular condyles.

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We previously identified the glucan-binding protein C gene, gbpC, solely involved in dextran-dependent aggregation (ddag) of Streptococcus mutans. Recently, we identified two gbpC gene homologues, gbpC and dbl, in Streptococcus sobrinus, and suggested that the dbl gene was very likely responsible for ddag of this species. However, homology searches with the gbpC or dbl genes against the incomplete TIGR S. sobrinus 6715 database suggested that this strain may have other gbpC homologues. PCR-based chromosomal walking from the gbpC and dbl genes revealed two additional homologous genes designated as gbpC2 and dblB. Proteins encoded by these genes exhibited alpha-1,6 glucan-binding activities. Therefore, gbpC2 and dblB are also logical candidates responsible for the ddag phenotype.

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