Ryuji Kodama

Nax sodium channel is expressed in non-myelinating Schwann cells and alveolar type II cells in mice

Na(x) is an extracellular sodium-level-sensitive sodium channel expressed in the circumventricular organs in the brain, essential loci for the sodium-level homeostasis in body fluids. Here, we examined the localization of Na(x) throughout the visceral organs at the cellular level. In visceral organs including lung, heart, intestine, bladder, kidney and tongue, a subset of Schwann cells within the peripheral nerve trunks were highly positive for Na(x). An electron microscopic study indicated that these Na(x)-positive cells were non-myelinating Schwann cells. In the lung, Na(x)-positive signals were also observed in the alveolar type II cells, which actively absorb sodium and water to aid gas exchange through the alveolar surface. It was thus suggested that the Na(x) sodium channel is involved in controlling the local extracellular sodium level through sodium absorption activity.

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.

Immunological relationships among embryonic and adult chicken pepsinogens: a study with monoclonal and polyclonal antibodies

To investigate the immunological relationships of pepsinogen isozymes present in embryonic and adult chicken proventriculi, we obtained monoclonal and polyclonal antibodies to these pepsinogens. Zymograms and immunoblots demonstrated that monoclonal antibody Y37 reacted with both embryonic and slow‐migrating adult pepsinogens, while polyclonal antibodies against embryonic pepsinogen and fast‐migrating adult pepsinogen were specific for these respective antigens. Shift from embryonic to adult‐type pepsinogen occurred at about the time of hatching and the localizations of embryonic and adult‐type pepsinogens within proventricular gland cells were found to differ by the indirect immunofluorescence method. Results with these antibodies revealed the immunological relations of these pepsinogens and the unique properties of embryonic chicken pepsinogen.

Ontogenesis of α2-adrenoceptor coupling with GTP-binding proteins in the rat telencephalon

Abstract: The ontogenesis of α2‐adrenoceptors and GTP‐binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese‐induced elevation of [3H]clonidine binding was increased in an age‐dependent manner but the guanosine 5′‐O‐(3‐thio)triphosphate‐induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age‐dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP‐ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39‐kDa proteins ADP‐ribosylated by pertussis toxin (Giα+ Goα) were increased with age and reached the adult level at day 12, whereas the 46‐kDa protein (Gsα) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against α‐ and β‐subunit of GTP‐binding proteins) demonstrated that the 39‐kDa α‐subunit of (Goα) and the 36‐kDa δ‐subunit of GTP‐binding protein (δ36) increased with postnatal age. In contrast, 35‐kDa δ‐subunit (δ35) did not change. From these results, it is suggested that the coupling activity of α‐adrenoceptor with GTP‐binding protein gradually develops in a manner parallel with the increase of α2‐adrenoceptor and pertussis toxin sensitive GTP‐binding proteins, Gi, and that α39β36γ may be related to the differentiation and/or growth of nerve cells in rat telencephalon.

Gene regulation and differentiation in vertebrate ocular tissues

Molecular biological techniques have contributed greatly to the study of vertebrate ocular tissues. The specification of ocular tissues has been shown to be closely related to the expression of transcription factors encoded by genes such as Pax6 and microphthalmia. Lens-specific expression of the delta 1-crystallin gene is controlled by factors, such as delta EF1, binding to its enhancer sequences. Retinal activity of the glucocorticoid hormone receptor is regulated by its binding with another transcription factor. Degeneration of photoreceptors in a retinal disease, retinitis pigmentosa, can be caused by the introduction of a mutated opsin gene into mice. In addition, the process of transdifferentiation in ocular tissues has been described at the level of gene expression.

Retinal differentiation from multipotential pineal cells of the embryonic quail

Pineal cells of the embryonic quail are multipotent stem cells which are able to differentiate in vitro into pigmented epithelial cells, lens cells and skeletal muscle fibers. Neuronal expression was added in this study in the repertory of differentiating potency of pineal cells. We used immunohistochemical methods to characterize neuronal properties with antibodies against serotonin, GABA, tyrosine hydroxylase and neuron-specific antigen (HPC-1) in addition to the enzyme histochemistry for acetylcholinesterase activity. Cells in the culture were found to be positively stained with these methods, suggesting that embryonic pineal cells are neuropotent to differentiate various types of neuronal cells. We have studied the culture conditions which favor increment of neuronal cells with extension of neuritic processes, and we have found that neuronal cells are maintained for quite a long period under suppressive conditions of DNA synthesis and under the effect of basic fibroblast growth factor (FGF). Suppression of DNA synthesis was achieved by the addition of aphidicolin, an inhibitor of DNA polymerase alpha, in the medium. Time lapse videograph revealed two different cell types participated in neurogenesis; a minor population of small round cells and a major one of flat epithelial cells. Since embryonic quail pineal cells have been shown to differentiate into two types of photoreceptors, the present results show wider retinal potency of cell differentiation by embryonic pineal cells. The cessation of DNA synthesis as well as growth factor(s) may be positively involved in the mechanisms of determination and differentiation of pineal neurons.

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I‐ or type IV‐coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti‐β1 integrin antibody was added to the PEC culture medium, a decrease of cell‐substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell‐substrate adhesion.

Density dependent growth of corneal endothelial cells cultured in vitro.

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.

Expression of the Retinal Pigmented Epithelial Cell‐Specific pP344 Gene during Development of the Chicken Eye and Identification of Its Product

In order to understand the transdifferentiation of the retinal pigmented epithelial cells (PECs) into the lens cells at the level of gene expression, a gene, tentatively called pP344 gene, was studied, because its expression appeared to be closely related with the differentiated state of PECs. We analyzed pP344 gene expression during chicken eye development by RT‐PCR and in situ hybridization and also characterized the pP344 protein using antipeptide antibodies. In addition to the previous observation that the transcript of pP344 gene is limited to the pigmented epithelium and not detected in the melanocytes, we show here that the transcript is limited to retinal PECs and is never observed in iris or ciliary PECs. The time course of expression level showed two peaks; the first peak occurred around the 10th day similarly to the expression of melanosome‐related genes, while the second peak occurred just after hatching when PECs had completely differentiated, suggesting that pP344 gene may be related to the function of fully differentiated PECs. Antisynthetic peptide antibodies detected pP344 protein in the culture medium of the PECs but not within the cells. Thus, we concluded that pP344 gene is specifically expressed by the retinal PEC and its product is a secreted protein.

Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle in avian retinal pigmented epithelial cells in vitro

SummaryThe dedifferentiated phenotype of pigmented epithelial cells in vitro is bipotential and is effected by environmental alterations mediated by the cell surface and associated cytoskeleton. We have begun an investigation into the role that contractile microfilaments play in maintaining cell contact and cell shape in retinal pigmented epithelial cells in vitro. In this paper, we report a structural analysis of the intersection of the circumferential microfilament bundle with the cell membrane of cultured pigmented epithelial cells from chick retina. Techniques of electron microscopy, including freezefracturing and deep-etching, reveal that microfilaments of this bundle associate with a junctional complex in the apical cell compartment and with membrane domains which are not components of the junction. Microfilaments link with the cell membrane either at their termini or along the membrane-apposed surface of the circumferential bundle. Furthermore, we report the immunocytochemical localization of filamin (a high molecular weight actin-binding protein, which forms fiber bundles and sheet-like structures when bound with Factin in solution) in the circumferential/microf