To identify the distribution of endogenous serum proteins in living mouse renal glomeruli under various hemodynamic conditions, we used the periodic acid-Schiff (PAS) and its fluorescence emission as a marker for the glomerular basement membrane (GBM). The immunostaining for collagen type IV was hardly observed without microwave treatment in specimens prepared by an “in vivo cryotechnique”. However, PAS staining and its fluorescence emission could be clearly visualized at the GBM with the “in vivo cryotechnique”. Under normotensive conditions, immunoreaction products of albumin and immunoglobulin G heavy and light chains (IgG(H+L)) were localized within glomerular capillary loops (GCL) but not colocalized with the PAS fluorescence emission of the GBM. Under heart-arrest conditions and with quick-freezing of resected tissues, albumin, IgG (H+L), immunoglobulin kappa light chain, and IgG1 heavy chain (IgG1) were immunolocalized within the GCL and mesangial areas, but only albumin and the kappa light chain were additionally immunolocalized in Bowman’s space, indicating their passage through the GBM. Under acute hypertensive conditions, both albumin and the kappa light chain, but not IgG1, were clearly immunolocalized along the GBM and in the Bowman’s space, indicating their increased passage through the GBM. The overlapping areas of PAS fluorescence emission and the albumin or kappa light chain appeared to be larger with quick-freezing and under the heart arrest or acute hypertensive conditions than under normal circulation, whereas those of PAS emission and IgG1 did not differ among these conditions. The serum proteins passing through the GBM were clearly visualized with the “in vivo cryotechnique”, immunofluorescence staining, and PAS fluorescence

View full abstract

Chick serum (CKS) contains factors that stimulate adipocytes in Meckel’s chondrocytes in vitro. In the present study, we analyzed levels of fatty acids in CKS, and further examined whether these had the potential to convert chondrocytes to adipocytes. Phenotypic changes were evaluated by light and electron microscopies, bromodeoxyuridine (BrdU) incorporation, triglyceride assays, and immunocytochemistry. We showed that CKS contained high levels of fatty acids, and a mixed medium containing 5 particular fatty acids inhibited DNA synthesis and the proliferation of chondrocytes as it facilitated their differentiation into adipocytes. The adipocytes produced were sudan-positive multilocular cells that morphologically and histochemically resembled adipocytes induced by the CKS-containing medium. Almost all lipid droplet-containing cells were positive for leptin and α-glycerophosphate dehydrogenase (GPDH), as evaluated by immunoperoxidase staining, and their triglyceride concentrations markedly increased during 4 to 6 days of culture. These results suggested that specific fatty acids in CKS are involved in the adipocytic conversion of Meckel’s chondrocytes.

View full abstract

Morphological reaction of sensory neurons of dorsal root ganglia after peripheral nerve transection was investigated by a nerve tracing method using E. coli lacZ (β-galactosidase) gene recombinant adenovirus. The sciatic nerve of the rat was transected and inoculated with the gene recombinant adenovirus from the cutting end of nerve fibers. The fixation was accomplished from one to six weeks after inoculation. A whole mount specimen was observed after the reaction in a X-galactocidase substrate. Newly formed sprouting processes of dorsal root ganglion (DRG) cells appeared, all of them sprouting from the primary segment of DRG cells. Developed branches were morphologically categorized in to two types: one was the “linear type” which showed diverged branches running straightly along the major axis of the DRG; the other was the “winding type” which exhibited a random running pattern to the original axons and wound and extended in all directions in dorsal root ganglia with many branches. Many of this type encircled other cell bodies and formed a ring-like structure. There was no difference in the size of cell bodies in either type or between the ring-like structure forming the cells and those cells encircled by them.

View full abstract

The present study investigated the expression and distribution of osteopontin in the mouse major salivary glands. The level of osteopontin expression in the mouse submandibular gland was higher (12.7-fold) than that in parotid and sublingual glands at the mRNA level. By Western blot analysis, intense positive bands were seen at the predicted molecular mass (about 55kDa) in all the major salivary glands, while an approximately 30kDa band of osteopontin was detected only in the submandibular gland. Indirect immunofluorescent and immuno-electron microscopy analyses demonstrated the localization of osteopontin in the luminal (apical) membranes of acinar cells in all the salivary glands. Osteopontin was also localized at the lumen of acini in the submandibular gland. These results suggest that the expression of osteopontin in the submandibular gland is different from that in the parotid and sublingual glands and that osteopontin may be degraded in the mouse submandibular gland.

View full abstract

Taste buds are multicellular receptor organs embedded in the lingual epithelium of vertebrates. Taste cells within these buds are modified epithelial cells as they lack axons and turnover rapidly throughout life, yet have neuronal properties enabling them to transduce taste stimuli and transmit this information to the nervous system. Taste cells are heterogeneous, comprising types I, II, III and basal cells, and are continually replaced during adult life, raising the question of how these different cells are generated. The molecular mechanisms governing taste cell differentiation are unknown, but the Notch signaling system has been implicated in this process based upon recent gene expression data. Here we investigate the expression in mature taste buds of Notch related transcription factors, Hes6 and Mash1, which are among the first genes expressed in embryonic taste buds. We further compare these patterns with those of immunocytochemical markers of discrete taste cell types. We find that Hes6 is expressed in a subset of basally located, possibly progenitor cells, yet is rarely coexpressed with taste cell markers. In contrast, Mash1 is detected in some basal cells and in the majority of differentiated type III taste cells, but never in type II cells. These data suggest a role for Notch signaling in taste cell differentiation in adult taste buds.

View full abstract