Expression of synaptotagmin 1 in the taste buds of rat gustatory papillae

Synapses between taste receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the central nervous system. The synaptic vesicle cycle at the synapses involves vesicle docking, priming, fusion, endocytosis, and recycling. Many kinds of synaptic vesicle proteins participate in synaptic vesicle cycles. One of these, synaptotagmin 1, binds Ca(2+) phospholipids with high affinity and plays a role in Ca(2+) regulated neurotransmitter release in the central and peripheral nervous systems. However, the expression patterns of synaptotagmin 1 in rat taste tissues have not been determined. We therefore examined the expression patterns of synaptotagmin 1 and several cell specific markers of type II and III cells in rat taste buds. RT-PCR assay showed that synaptotagmin 1 mRNA was expressed in circumvallate papillae. In fungiform, foliate, and circumvallate papillae, the antibody against synaptotagmin 1 yielded the labeling of a subset of taste bud cells and intra- and subgemmal nerve processes. Double labeled experiments showed that synaptotagmin 1 positive cells co-expressed type III cell markers, PGP 9.5, and NCAM. Intragemmal nerve processes positive for synaptotagmin 1 co-expressed PGP 9.5. Conversely, all synaptotagmin 1 expressing cells did not co-expressed type II cell markers, PLCbeta2, or gustducin. These results show that synaptotagmin 1 may play some regulatory roles in vesicle membrane fusion events with the plasma membrane at the synapses of type III cells in rat taste buds.     

Bcl11b is required for differentiation and survival of alphabeta T lymphocytes

The gene Bcl11b, which encodes zinc finger proteins, and its paralog, Bcl11a, are associated with immune-system malignancies. We have generated Bcl11b-deficient mice that show a block at the CD4-CD8- double-negative stage of thymocyte development without any impairment in cells of B- or gammadelta T cell lineages. The Bcl11b-/- thymocytes showed unsuccessful recombination of V(beta) to D(beta) and lacked the pre-T cell receptor (TCR) complex on the cell surface, owing to the absence of Tcrb mRNA expression. In addition, we saw profound apoptosis in the thymus of neonatal Bcl11b-/- mice. These results suggest that Bcl11b is a key regulator of both differentiation and survival during thymocyte development.     

Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.     

Catalase, a Specific Antigen in the Feces of Human Subjects Infected with Helicobacter pylori

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C₅₀ chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.     

Three-dimensional cytoarchitecture of the media of arteriovenous anastomoses in the rabbit ear

Arteriovenous anastomoses in the rabbit ear were examined with scanning electron microscopy to elucidate the structural differentiation of the media of the shunt. Arterial, intermediate, and venous segments in the shunt and two layers of the media in the intermediate segment were differentiated based on cell shape and cell organization. In the arterial segment, smooth muscle cells were spindle-shaped, either elongated or short, with a few branches, and were arranged circularly or diagonally with respect to the vessel's long axis. There were also stellate muscle cells with radiating processes. In the intermediate segment, the smooth muscle cells of the outer layer of the media were also arranged circularly and resembled the elongated cells in the arterial segments, but they were more irregular in shape and had more processes than those of the arterial segment. The epithelioid cells of the inner layer of the media were oval or polygonal and oriented irregularly with respect to the vessel's long axis, clustering to form longitudinal plicae. The smooth muscle cells of the venous segment were flat with many lateral processes and formed a thin, discontinuous layer. The smooth muscle cells in the arterial segment and those of the outer layer of the intermediate segment exhibited a highly rugged surface texture, indicating their strong contractility; the epithelioid cells and the smooth muscle cells in the venous segment exhibited a generally smooth surface, indicating less contractility. The intermediate segments were supplied with a dense nerve plexus. The intermediate segments, therefore, may be actively involved in the regulation of blood flow under neuronal influence.     

Comparison of mutagenic potentials and mutation spectra of benzene metabolites using supF shuttle vectors in human cells

Benzene is a human leukemogen and the metabolites are thought to be deeply involved in benzene leukemogenesis. In a previous study we reported the molecular analysis of p-benzoquinone (p-BQ) mutagenesis by using a supF shuttle vector plasmid and here we report the mutagenesis of the other metabolites, hydroquinone (HQ) and trans, trans-muconaldehyde (MUC). HQ is a precursor of p-BQ and MUC is produced by a ring-opening metabolic pathway. We found that the HQ redox cycle produced an oxidative lesion in plasmid DNA and significant differences among the mutagenic potentials of MUC, HQ and p-BQ. HQ has stronger mutagenicity than the others. It is about 20 and 600 times stronger than p-BQ and MUC, respectively. Furthermore, we found notable differences in each mutational feature. The MUC mutational type was characterized by a high frequency of tandem base substitutions that could be due to crosslinks produced by its aldehyde moieties, while HQ was characterized by frequent deletion. This HQ feature is the same as in vivo benezene mutagenesis of Big Blue mice reported by Provost et al. in 1996 and is also quite similar to a hydrogen peroxide mutational feature. Therefore, we presume that HQ and reactive oxygen species may play an important role in benzene carcinogenesis.     

Vesicles in the subacrosomal space and partial diaphragms in the subacrosomal nuclear envelope of round spermatids of a rat injected intravenously with gold labeled-testosterone-bovine serum albumin conjugate: vesicular trafficking from acrosome to nucleus

Colloidal gold labeled-testosterone-bovine serum albumin conjugate (testosterone-BSA-gold) injected into the vascular system of rats is taken up by endocytosis into round spermatids. Based on observation of silver deposits indicating testosterone-BSA-gold with silver enhancement, we have suggested that testosterone-BSA-gold enters the nuclei through not only the postacrosomal nuclear envelope but also the subacrosomal nuclear envelope (SNE) via the acrosome (Nishimura and Nakano, 1997). However, it was unclear how testosterone-BSA-gold in the acrosome entered the nucleoplasm. Spermatids showing silver deposits on the subacrosomal space were observed under electron microscope without silver enhancement, to clarify the courses of translocation. In the spermatids, vesicles with the gold particles were seen in the subacrosomal space. Some of the vesicles were in contact with the SNE. A part of the outer nuclear membrane projected into the space. Furthermore, local single-bilayer nuclear membranes, which seemed to partially lack nuclear lamina, were present in the SNE. These results indicate the possibility that the vesicles mediate the transport of testosterone-BSA-gold from acrosome to nucleus, and that the vesicle membrane fuses with not only the outer nuclear membrane but also a shared bilayer in the SNE.     

Rapid Detection of Species of the Opportunistic Yeast Trichosporon by PCR

Trichosporon species are opportunistic pathogens, associated with a high mortality rate in immunocompromised patients. Oligonucleotide primers were used to amplify a 170-bp fragment of small-subunit ribosomal DNA of all species in the genus Trichosporon by PCR. The primers amplify DNAs of all species in the genus Trichosporon, including six causative agents of trichosporonosis. DNAs of other medically important yeasts, such as Candida albicans and Cryptococcus neoformans, are not amplified by this detection system.     

DNA analysis of two patients with a non-fluorescent y chromosome

Summary Results of DNA study on two patients of gonadal dysgenesis with a 45,X/46,X,Ynf (non-fluorescent Y chromosome) karyotype are described. In one patient who developed gonadoblastoma, all 12 loci on the non-fluorescent part of Yq were detected. Another patient did not have gonadoblastoma at 20 years, and only the proximal 6 loci out of 12 were detected.