Douk Ho Hwang

Immunohistochemical study of the distribution of sodium-dependent vitamin C transporters in adult rat brain

Sodium‐dependent vitamin C transporters (SVCTs) is known to transport the reduced form of ascorbic acid into the cell, whereas the oxidized form of vitamin C (VC) is moved through a facilitative sugar transporter, such as glucose transporter (GLUT). With regard to the distribution of SVCT1 and ‐2 within the various organs, they were reported to be expressed in different types of cells. Especially in the central nervous system, only SVCT2 mRNA was expressed mainly in neurons and some types of neuroglial cells. However, data on the expression of SVCT proteins in the brain are scant. Therefore, we tried to develop comprehensive data on the distribution of SVCT proteins in adult rat brain by using immunohistochemical techniques for the first time. In our study, SVCT2 immunoreactivities (IRs) were intensely localized in the neurons of cerebral cortex, hippocampus, and Purkinje cells of cerebellum, and much weaker SVCT2 IRs were found in the other brain regions. Judging from double‐immunohistochemical data, most of the cells expressing SVCT2 IRs were likely to be neurons or microglia, even though the cells in choroids plexus or ependymal cells around the ventricles also exhibited SVCT2 IRs. Complete mapping of the distribution of SVCT2 IRs was available by using a semiquantitative method. The subcellular localization of SVCT proteins is necessary for understanding the exact role of the protein, so the current overall mapping of SVCT IRs in the rat brain could be the basis for further studies on related subjects.

Immunocytochemical localization of neuronal and inducible nitric oxide synthase in the retina of zebrafish, Brachydanio rerio.

Several previous studies have revealed the distribution of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in the retina of various species. However, nothing has been reported on the nNOS and iNOS expression in zebrafish retina, even though it has been used as an effective model for developmental studies. In this study on nNOS and iNOS immunoreactivity (IR) in the zebrafish retina, iNOS-IR signals were detected in the ganglion cell layer (GC), the inner plexiform layer (IP), the outer plexiform layer (OP) and the photoreceptor layer (PR). nNOS-IR signals were also intensely detected in the GC, IP, OP and PR. These signals were very similar to the iNOS-IR signals, except for some minor differences in relative intensities. This could be explained by the speculation that the synthesis of nitric oxide by iNOS may represent a compensatory mechanism in the absence of nNOS. We described for the first time the distribution of nNOS and iNOS-IR cells in zebrafish retina. This could provide the basis for further study on the nNOS and iNOS properties of zebrafish retina.

Distribution of transferrin binding protein immunoreactivity in the chicken central and peripheral nervous systems

Transferrin binding protein (TfBP) is a glycoprotein originally purified from chicken oviduct that exhibits transferrin binding activity. Recent work has shown that TfBP is a post‐translationally modified form of the heat shock protein (HSP108), the avian homologue of a glucose regulated protein, GRP94. The function of this protein, however, has not yet been clearly defined. Antiserum to TfBP was found to selectively stain oligodendrocytes of the avian brain. In this study, we further describe these oligodendrocytes and other cell types positive to anti‐TfBP in the chick nervous system. In accordance with previous studies, the most prominent cell type that labels with antiserum to TfBP is the oligodendrocyte. At the electron microscopic level, the immunoreactive product is confined to the perinuclear cytoplasm and fine processes of the oligodendrocytes, whereas myelin and axoplasm are devoid of staining. The immunoreactive product is found both in the cytoplasmic matrix and bound to the endoplasmic reticulum and plasma membrane, suggesting that TfBP may have properties of both a soluble and an integral membrane protein. There is great variability in the number of TfBP‐oligodendrocytes in different areas of the central nervous system (CNS); large numbers of cells are associated with the white matter regions and are found in the myelinated tracts, whereas few cells are present in the gray matter regions. In the retina, TfBP is localized specifically in the cells that are morphologically oligodendrocytic and is present in the optic nerve fiber layer and the ganglion cell layer. Obvious staining is also seen in the Bergmann glial cells of the cerebellum and in the Schwann cells of the sciatic nerve. Furthermore, the choroid plexus cells similarly exhibit a strong reaction. The association of TfBP in these specific cell types responsible for myelination and sequestering iron and transferrin implies that TfBP may be involved in myelination and iron metabolism of the chick nervous system, perhaps through a role in transferrin concentration in these cells. A putative role of TfBP, as HSP108, is considered. J. Comp. Neurol. 260‐271, 1997.

Oligodendroglia in the avian retina: immunocytochemical demonstration in the adult bird.

Immunohistochemical techniques were used in conjunction with an avian‐specific probe for oligodendrocyte (OLG) marker, the antibody for transferrin binding protein (TfBP), to study the characteristics and distribution of OLGs in the retina of chickens and quails. For comparison, other antibodies such as myelin basic protein, Rip, and those for labeling Müller cells and microglia were used. A large population of OLGs was found to be distributed throughout the retina, with the distinct pattern of a central‐to‐peripheral gradient. It was possible to detect a spectrum of OLG morphology that bore a resemblance to the subtype of the mammalian central nervous system. In addition to these mature OLGs, limited numbers of TfBP‐positive (TfBP+) cells with the morphology of immature OLGs were found in the immediate vicinity of the optic head. The majority of OLGs appeared in the ganglion cell layer throughout the retina, whereas OLGs in the nerve fiber layer were seen mainly in the central zone of the retina, near the optic nerve head. Double‐labeling experiments showed that OLGs were associated with myelin only in the central region, where the majority of retinal OLGs occurred, but not toward the periphery of the retina. The present study is the first comprehensive analysis of the morphological features and spatial distribution of OLGs in the adult avian retina and provides in vivo evidence for the existence of a substantial population of both mature and immature OLGs in the retina of adult birds. The putative functions of TfBP+ OLGs including myelination and the tropic role of the ganglion cells are discussed in conjunction with the physical properties of TfBP and structural characteristics of the avascular retina of birds. J. Neurosci. Res. 65:173–183, 2001.

Localization of transferrin binding protein in relation to iron, ferritin, and transferrin receptors in the chicken cerebellum

We have demonstrated that transferrin binding protein (TfBP), ferritin, and iron, are specifically localized in Bergmann glia, while the transferrin receptor is confined to Purkinje cells in the chicken cerebellum. The results of this study suggest that Bergmann glia have previously undescribed functions related to iron regulation such as sequestration of iron and the maintenance of iron homeostasis in the cerebellum.

CM1, a possible novel activation molecule on human lymphocytes

CM1 (centrocyte/-blast marker 1) defined by a mAb developed against concanavalin-A activated PBMC, is expressed specifically on some tonsillar germinal center (GC) B cells. In single flow cytometric analysis, the bone marrow did not express these molecules nor did the PBMC or the thymocytes. The peripheral B lymphocytes showed more than 90% positive, while the peripheral T lymphocytes showed approximately 60% positive at 48 h after activation by PMA/ionomycin, respectively. A western blot analysis and an immunoprecipitation for CM1 showed a band at 70 kDa. Cross-linking of CM1 with anti-CM1 mAb induced apoptosis of the GC B cells (CD38(+)IgD(-)). Immunohistochemical staining revealed that the CM1 molecule is distributed over the entire area except the proximal dark zone of the tonsillar germinal centers. These results suggest that the CM1 molecule might be involved in differentiation of the germinal center B cells as one of the novel centrocyte markers.

Localization of bcl-2 mRNA in the rabbit central nervous system

In this study, we demonstrated bcl-2 expression in the rabbit brain using in situ hybridization and compared the results with the studies done on the other animals. Although the bcl-2 expressions of the rabbit were generally similar to those of the other animals, some differences were also found; the cells in molecular and Purkinje cell layers of the cerebellum, which were bcl-2 negative in the others, showed intense bcl-2 positive signals, while the ependymal cells, arachnoid villi and granular layer, which were positive in the other animals, were not well stained with bcl-2 riboprobes in the rabbit brain. Our findings clearly showed the similarities and differences of rabbit bcl-2 mRNA as compared with the other species for the first time, and provided the basis of further study on the properties of bcl-2 in this species.

Are substance P neurons of the paraventricular nucleus related to the osmotic regulation in the Mongolian gerbil

In recent years, the gerbil has been used as an experimental animal for study osmotic regulation, because of its inherent high degree of water re-uptake in kidney. Many evidences to explain this characteristic accumulated on the kidney level, it do not, however, manifest what concerns on hypothalmo-hypophyseal level. In this study, we have focused on the difference between the colchicine treated gerbil and rat in distributions of substance P (SP), which is known to have antidiuretic property. Unlike rat, in which a few SP+ neuron is present, SP+ neurons were abundantly observed in the paraventricular nucleus (PVN) of the gerbil. Furthermore SP+ cells in PVN were manifested in dehydrated gerbils, in spite of non-colchicine treatment. Therefore, we suggest that the abundant SP+ neurons in PVN may be a clue to address the neuroendocrinal mechanism concerning the high degree of osmotic regulation in this animal.

Activation of microglia in kainic acid induced rat retinal apoptosis

We applied a variety of methods to follow the course of kainic acid (KA) induced retinal apoptosis, especially with regard to the spatial and temporal aspects. At 24 h after KA injection, a massive cell increase, which showed terminal transferase-mediated dUTP nick-end-labeling technique positive signals, was observed in all of the retinal layers, with the exception of the outer nuclear and photoreceptor layers. Electron microscopy further confirmed that these cells might be apoptotic body ingesting phagocytes, whose function seemed to correlate with bcl-2 mRNA up-regulation. When histochemical studies were performed to determine the cellular identity of the phagocytes, the microglia were thought to be the one and only type of phagocytes involved in the KA-induced retinal apoptosis. In conclusion, we demonstrated that after KA injection, microglia were the only phagocytes to participate in clearing apoptotic debris from the inner retinal layers, and that their function might correlate with the change in expression of the bcl-2 gene family.