Fine structure and lectin histochemistry of the apical surface of the free neuromast of Lampetra japonica

The surface coat, ciliary process, and microvilli of the lamprey neuromast were examined with electron microscopy after tannic acid prefixation and lectin histochemistry. The neuromast was found to exist in the form of a dermal mound with a furrow in the middle. On the bottom of the furrow, the hair cell was characterized by a kinocilium and 15–20 stereocilia, arranged along the longitudinal axis of the furrow. Spanning structures were demonstrated between the kinocilium and stereocilia as well as between stereocilia. The surface coat, enhanced by tannic acid prefixation, was particularly rich over the surface of the supporting cell; by contrast, it was thin over the hair cell. Some lectins (PNA, GS-I, SBA, WGA) showed affinity to the surface coat of the supporting cell as well as the hair cell, and the others (RCA-I, MPA, ConA) showed affinity only to the supporting cell. These differences in the structure and affinities of the surface coat suggest an extracellular milieu highly specialized for the hair cell in this particular form of the mechanoreceptor.     

New anatomical method of grain angles measurement using confocal microscopy and image cross-correlation

Some tropical trees with indistinct growth rings have a distinct interlocked grain that reveals their internal growth rhythm. To determine their growth rhythm, it is necessary to accurately measure the wood grain angle. The usual methods for grain angle measurement are radial splitting using wood disks, which occasionally provides inaccurate data, and serial tangential sectioning, which requires preparation and analysis of many sections. The present report proposes an easier but accurate method to measure grain angle using a single xylem transverse section. A confocal microscope was used to obtain two optical sections of different depths from a transverse section of a 7-year-old Hopea odorata Roxb. The tangential lag between the optical images was then calculated using image cross-correlation and transformed into grain angle. Radially consecutive sampling revealed distinct radial fluctuations in the grain angle. The fluctuation data were compared to data obtained by radial splitting and serial tangential sectioning. There was a strong correlation between grain angle using the three methods. In the region close to the cambium, however, the present method revealed an abrupt change in the grain angle, although radial splitting showed a smooth undulation throughout the radius. Using the present method, the analysis of a radial range of 5 cm required a single transverse section compared to 1,000 tangential sections 50-m thick. In conclusion, the present method using a single transverse section, confocal microscopy, and image cross-correlation analysis provides more accurate data than radial splitting, and is less time-consuming than serial tangential sectioning.     

Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.     

A novel action of activin A: stimulation of insulin secretion in rat pancreatic islets

The present study was conducted to examine an action of activin A on insulin secretion from rat pancreatic islets. In a batch incubation system, activin A stimulated insulin secretion in a dose-dependent manner at concentrations higher than 1 nM. Furthermore, activin A greatly potentiated glucose-induced insulin release. When islets were perifused with 1 nM activin A, insulin secretion was barely affected in this system. However, the insulin response to 16.7 mM glucose was greatly enhanced. Both the first and second phases of insulin response were enhanced by 1 nM activin A. These results indicate that, in addition to its known actions on pituitary-gonadal and hematopoietic systems, activin A modulates the function of pancreatic islets and stimulates insulin secretion.     

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.     

Theanine,gamma-glutamylethylamide,is metabolized by renal phosphate-independent glutaminase

The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for L-glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that gamma-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and L-glutamine, in view of relative activity and K(m) value. It was suggested that gamma-glutamyl moiety in theanine molecule was transferred to form gamma-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of gamma-glutamyl transpeptidase reaction to other peptides in vivo.     

Mutations affecting somite formation in the Medaka (Oryzias latipes)

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.     

The efficacy of a herbal medicine (Mao-to) in combination with intravenous natural interferon-beta for patients with chronic hepatitis C, genotype 1b and high viral load: a pilot study.

Patients with chronic hepatitis C, with a high serum viral load (> or = 1 Meq/ml) and genotype 1b seem to be resistant to interferon (IFN) therapy. To evaluate the efficacy of a herbal medicine (Mao-to) in combination with natural IFN-beta for the treatment of these patients, eighteen Japanese patients were enrolled in this study. Every patient received 6 million units (MU) of IFN-beta intravenously daily for 8 weeks. Mao-to was given orally 3-4 times a day during the IFN-beta administration, Sixteen of the 18 patients (89%) became negative for serum HCV RNA at the end of treatment, but only 2 of them (11%) remained negative for the virus RNA at 6 months of follow-up. Serum ALT levels normalized in 17 patients (94%) at 2 weeks of follow-up after the cessation of therapy, and 11 patients (61%) retained normal ALT levels for more than 6 months of follow-up. This rate of biochemical response was high as compared with that of therapy with IFN-beta alone (19%) in the largest IFN-beta trial in Japan. Serum hyaluronic acid levels were decreased significantly from 147.0 +/- 110.5 ng/ml to 77.4 +/- 67.4 ng/ml in the sustained biochemical response group (P = 0.003). None of the patients needed to interrupt therapy because of side effects of IFN-beta. Thus, Mao-to administration together with IFN-beta treatment could increase the sustained biochemical response rate, and reduce liver fibrosis.