Fine structure of soft and hard tissues involved in eye migration in metamorphosing Japanese flounder (Paralichthys olivaceus)

The body of a Japanese flounder (Paralichthys olivaceus) changes from a symmetrical to an asymmetrical form during metamorphosis. To obtain detailed information on the mechanisms of the migration of the right eye to the left side, soft and hard tissues in the head of larval flounders were examined using transmission electron microscopy (TEM). Retrorbital vesicles (Rvs) are pairs of sac-like structures under the eyes. It has been suggested that the asymmetrical development of Rvs, with the right (blind) one being bigger than the left, is the driving force behind eye migration. The present study revealed that the ultrastructure of the Rv sheath is quite similar to that of a lymphatic capillary. Thus, it is possible that the Rv is a part of the lymph system, and is probably related to the secondary vascular system in teleosts. If we assume that the Rv sheath has a high permeability to liquid, similar to lymphatic capillaries, it is not plausible that the active expansion of the Rv pushes the eyeball. On the other hand, the pseudomesial bar (Pb) is a bone that is unique to flounders and is present only on the right (blind) side. At the beginning of eye migration, an aggregation of fibroblast-like cells is observed in the dermis under the right eye, where the Pb will subsequently be formed. These cells have a well-developed rough endoplasmic reticulum (rER) and mitochondria, and are probably responsible for formation of the thick layers of collagen fibrils around them. Since it is unlikely that the active expansion of the Rv causes eye migration, the role played by the Pb and its rudiment becomes more significant in right eye migration in the Japanese flounder becomes more significant.     

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.     

Development of the articular cavity in the rat temporomandibular joint with special reference to the behavior of endothelial cells and macrophages

Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation; the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively.     

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.     

Characterization of a G14 equine rotavirus (strain CH3) isolated in Japan

Summary Antigenic and genomic properties of equine rotavirus strain CH3 isolated in Japan were studied by cross-neutralization tests and nucleotide sequence determination of the VP4 and VP7 genes. It was shown that the strain CH3 belongs to G14 and shares VP4 genotype with strain H2.The nucleotide sequence data reported in this paper appear in the DDBJ, EMBL and GeneBank nucleotide sequence detabases under the accession numbers D25228 (VP4 of strain CH3) and D25229 (VP7 of strain CH3).     

Immunocytochemical demonstration of heat shock protein 25 in the rat temporomandibular joint

The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp25 might be involved in the inhibition of apoptosis of those cells.     

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.     

Agarose for a bioartificial pancreas.

Islets were encapsulated into 5% concentration agarose microbeads. The effect of microencapsulation on islet allograft survivals was determined using a streptozotocin-induced diabetic (STZ) mouse and a nonobese diabetic (NOD) mouse as recipients. All five STZ BALB/c mice receiving microencapsulated islets (C57BL/6) maintained normoglycemia indefinitely. When NOD mice were used as recipients of the bioartificial pancreas, four of five grafts (islets from C3H/He) functioned for more than 80 d. Two of five NOD mice maintained normoglycemia until animals were sacrificed at 102 and 192 postoperative d. Microbeads made of commercially available agarose can effectively prolong alloislets functioning in the STZ-diabetic mouse and even in the NOD mouse (animal model of human type I diabetes) without the use of any immunosuppressive drug.