Kazushige Hirosawa

Defective degranulation and calcium mobilization of bone-marrow derived mast cells from Xid and Btk-deficient mice.

Regulation of adhesion and degranulation of mast cells plays an important role in allergy and inflammation. We investigated a possible role of Brutons tyrosine kinase (Btk) in the regulation of adhesion and degranulation by using bone marrow-derived mast cells from X-linked immunodeficiency (Xid) and Btk-deficient mice. Cross-linking of the high affinity IgE receptor (Fc epsilonRI) and steel factor (SLF) induced indistinguishable adhesive responses of mast cells to fibronectin in kinetics, and these adhesive responses were comparable among wild type, Xid, and Btk-deficient mast cells. Cross-linking of Fc epsilonRI, but not SLF triggered degranulation of bone marrow-derived mast cells. However, Fc epsilonRI-induced degranulation was impaired in Xid and Btk-deficient mast cells. Calcium influx induced by Fc epsilonRI cross-linking and SLF were also reduced in Xid and Btk-deficient mast cells. Degranulation and calcium influx were reduced more severely in Btk-deficient than in Xid mast cells. Consistently, cross-linking Fc epsilonRI and SLF augmented Btk kinase activities transiently. Inositol triphosphate (IP3) production was also severely reduced in Btk-deficient mast cells, indicating Btk play a critical role of Fc epsilonRI-induced IP3 production. The differential sensitivity of wortmannin on calcium influx in wild type and Xid mast cells suggested that the activation of phosphatidylinositol 3 kinase (PI 3-kinase) was required in calcium influx. Furthermore, abnormal secretory granules with translucent contents and variable in size were observed both in Xid and Btk-deficient mast cells. Our study demonstrated a critical role of Btk in regulating intracellular calcium and granule exocytosis.

Electron microscopic studies on pigment granules in the substantia nigra and locus coeruleus of the Japanese monkey (Macaca fuscata yakui)

SummaryThe so-called neuromelanin pigments in the substantia nigra and locus coeruleus ofMacaca fuscata yakui were studied electron microscopically and cytochemically. Eight Japanese monkeys were perfused with buffered 4% formaldehyde solutions. The materials were post-fixed in buffered 2% osmium tetroxide solutions for 2 hours or immersed in an incubation medium for acid phosphatase before post-osmification. They were dehydrated and embedded in Epon mixture.The neuromelanin pigment granules measure up to 1 μ in diameter and consist of three structural components which are similar to those of lipofuscin except that the former contain electron dense coarse patches. A conspicuous round and homogenous inclusion body without any crystalline structure occurs in the mitochondrial matrix.The probable process of neuromelanin formation is discussed, as well as the functional significance of intra-mitochondrial inclusions and acid phosphatase activity. It is suggested that neuromelanin pigments originate from lysosomes, in which altered catecholamines accumulate as coarse patches.

Structure of the subrhabdomeric cisternae in the photoreceptor cells of Drosophila melanogaster

SummaryThe structure of subrhabdomeric cisternae (SRC) and related structures in the photoreceptor cells (retinular cells) ofDrosophila melanogaster in normal flies and visual mutants were compared by electron microscopic observation of semithin sections of osmium-impregnated specimens. The three-dimensional organization of SRC and the other cell organelles was demonstrated by stereoscopy. Both light- and dark-adapted normal retinular cells contained elaborate networks of anastomosing tubules of SRC immediately beneath the rhabdomeres. Tubules connecting the SRC and rough endoplasmic reticulum were frequently seen. The SRC were absent from the retinular cells ofrdgAKS60 whose rhabdomeres degenerate gradually after eclosion. Instead, numerous smooth vesicles were observed in the subrhabdomeric regions. InrdgBEE170, in which rhabdomere degeneration is light dependent, the SRC appeared normal in the dark-adapted flies. But their SRC gradually disintegrated after exposure to light. InnorpASB37, whose rhabdomeres are small but do not degenerate, SRC appeared normal. These results suggest that the SRC is a significant structure for the maintenance of the structure of photoreceptive membrane in the retinular cells ofDrosophila.

Differentiation and morphogenesis in pellet cultures of developing rat retinal cells

We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461–467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell‐type‐specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Müller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate. J. Comp. Neurol. 377:341–350, 1997.

GLUT2 expression in the rat retina: localization at the apical ends of Müller cells.

In order to understand the molecular basis of glucose regulation supporting visual function, this study examined the presence of GLUT2, a facilitated-diffusion glucose transporter isoform, and delineated its localization in the rat retina. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of GLUT2 mRNA, and immunoblot analysis using polyclonal antibody specific to rat GLUT2 revealed a band at a molecular weight of approximately 60 kDa, indicating the presence of GLUT2 protein in the rat retina. Fluorescence and electron microscopy localized GLUT2 expression to the apical ends of Müller cells that face the inter-photoreceptor space. These findings suggest that GLUT2 on Müller cells may control intra-retinal glucose homeostasis by performing both anterior and posterior glucose transport within the rat retina. This is the first study to provide evidence that GLUT2 is present in the mammalian central nervous system and indicates that GLUT2 may have local glucose homeostatic functions within the retina in addition to its role in the regulation of systemic blood glucose level.

Structure of retinular cells in a Drosophila melanogaster visual mutant, rdgA, at early stages of degeneration

SummaryA Drosophila visual mutant rdgA has photoreceptive cells which degenerate gradually after eclosion. Fine structure of the retinular cells of rdgAKS60and rdgAK014 was studied during early stages of degeneration to determine the initial morphological defects. The retinular cells of these two alleles showed the following structural abnormality within 1 day after eclosion: (1) rhabdomeres were small and irregular in shape; (2) cisternae of the rough endoplasmic reticulum were more numerous than those in normal retinular cells; (3) submicrovillar cisternae were absent; and (4) lysosomes were fewer than normal. Three-dimensional reconstruction of serial sections of the ommatidia showed that the degeneration of mutant rhabdomeres proceeds more rapidly in regions remote from the nuclei. These results suggest that the process of turnover of rhabdomeric microvilli is abnormal in rdgA. We also confirmed an increase of lysosomes and destruction of cellular organelles, as reported by previous investigators at more advanced stages of degeneration.

LOCALIZATION AND ONTOGENY OF GLUT3 EXPRESSION IN THE RAT RETINA

This study investigates the presence, localization, and developmental expression of a neuron-specific facilitated-diffusion glucose transporter, GLUT3, in the rat retina so as to elucidate molecular mechanisms regulating glucose homeostasis in support of the visual function. Immunoblot analysis using anti-GLUT3 antibody (ALM3-C) revealed the presence of GLUT3 as a heterogeneously glycosylated protein with an average molecular weight of approximately 44 kDa. Although immunofluorescence staining showed it to be localized primarily in the inner and outer plexiform layers, some of the cell bodies in the inner nuclear layer also showed weak immunoreactivity. Immunoblot analysis of developing rat retinal tissues revealed the presence of the GLUT3 protein as early as embryonic day 15 (E15), and immunofluorescence staining revealed its expression in the inner plexiform layer near the time of birth and in the outer plexiform layer at postnatal day 14 (P14), i.e., when the eyes normally open and retinal activity commences. The proteins abundance remained at a relatively low level during the embryonic stages and up until the end of the first postnatal week (P7), though a transient increase was confirmed to occur at E18. From P13, however, the abundance steadily increased, rapidly reaching the adult level at P24. Based on these observations, we hypothesize that GLUT3 is expressed in some subsets of retinal neurons, being preferentially abundant in their neuronal processes, and that its ontogeny is closely associated with morphological and functional development of the retina. As such, this suggests that GLUT3 plays some important role(s) in the retina where glucose metabolism is essential.

Studies on the structure of ocellar photoreceptor cells of Drosophila melanogaster with special reference to subrhabdomeric cisternae

Abstract.We studied the structure of ocellar photoreceptor cells of Drosophila melanogaster, particularly the subrhabdomeric cisternae which our previous studies have shown to be essential structures for turnover of photoreceptive membranes in compound eyes. Each ocellus contained elongated photoreceptor cells with rhabdomeres positioned distally. In the subrhabdomeric regions, endocytotic invaginations were frequently observed, suggesting active turnover of photoreceptive membranes. In the vicinity of the photoreceptive microvilli, membranous structures similar to the subrhabdomeric cisternae in compound eyes were observed. These membranous structures were immunopositive for the rdgB protein, a phosphatidylinositol transfer protein that is localized to the subrhabdomeric cisternae in compound eyes. The ocellar photoreceptor cells of the retinal degeneration mutants (rdgA,B) were also studied. In these mutants, retinal degeneration has been reported to start, in compound eyes, with the disappearance of the subrhabdomeric cisternae. We found that the ocellar subrhabdomeric cisternae also disappear during the initial stage of retinal degeneration. From these observations, we conclude that the mechanism of photoreceptive membrane turnover in ocellar photoreceptor cells involves the rdgB and probably the rdgA proteins which are associated with subrhabdomeric cisternae, as is the case for photoreceptive membrane turnover in compound eyes.

Electron microscopic observations on the vomeronasal sensory epithelium of a crotaline snake, Trimeresurus flavoviridis

The vomeronasal sensory epithelium of a crotaline snake, Trimeresurus flavoviridis, was shown to consist of a superficial supporting cell layer and an underlying sensory cell layer composed of columns of sensory cells. The supporting cell layer consists of both supporting cells and dendrites of the underlying sensory neurons. The apical regions of sensory cell dendrites contain numerous microtubules, many elongated mitochondria, centrioles, and electron‐dense bodies. The dendrites terminate as dendritic knobs from which microvilli project into the vomeronasal lumen. Smooth vesicles are abundant in the dendritic terminals and their vicinity. Supporting cells also bear microvilli, and these cells contain large electron‐opaque granules and dense vesicles near their free surfaces. Cytoplasmic extensions of the supporting cells form a meshwork which separates dendrxites from each other in the vicinity of the luminal surface. The meshwork becomes obliterated in the infranuclear region of each supporting cell. Bipolar‐shaped sensory cells with lightly stained round nuclei contain the characteristic cell organelles of neurons and are thought to be sensory neurons. These cells are especially characterized by well‐developed lamellae of rough endoplasmic reticulum and extensive arrays of smooth endoplasmic reticulum. The perikarya of cells located in the apical region of the cell columns tend to contain larger amounts of smooth endoplasmic reticulum and lipofuscin granules than the perikarya of cells located in lower regions. Undifferentiated cells are found in the basal region of the columns. Satellite cells form the framework of the columns and are also found among neuronal elements.

Monoclonal antibodies which recognize endoplasmic reticulum in the retinal pigment epithelium

Balb/c mice were immunized with dissociated chick retinal pigment epithelial cells and monoclonal antibodies prepared. Retinas of chick, bovine, rabbit, rat and mouse were examined with the monoclonal antibodies by immunofluorescence microscopy. Among the antibodies obtained, two (S5D8 and S5H8) stained the cytoplasm of epithelial cells of the animals examined in finely granular pattern and recognized a 63-kDa protein in retinal pigment epithelial cells. These antibodies, however, did not react with the rest of the chick eye (cornea, iris, ciliary body, lens and sclera) nor with extraocular tissues (liver, kidney, intestine, brain, testis, adrenal gland, heart, gizzard and skeletal muscle). Immunoelectron microscopy revealed that the antigen was localized in the endoplasmic reticulum of retinal pigment epithelial cells.