Sumio Nakamura

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

SummaryTo investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.

Immunocytochemical localization of the allergenic proteins in the pollen of Cryptomeria japonica

Applying an immunocytochemical method, a localization of the protein Cry j I in the Cryptomeria japonica pollen, which is the major allergen responsible for Japanese cedar pollinosis, is investigated with the monoclonal and polyclonal antibodies produced from the protein. The protein that reacts to the polyclonal antibody localizes on the sexine, nexine, between nexine and intine layers, orbicles, cell wall of a generative cell, Golgi body and Golgi vesicles. The allergenic protein contained in the exine and orbicles of Japanese cedar pollen can diffuse or dissolve easily from there into the mucus covering of the eye and nose, causing a response in less than 1 min after exposure. Since the orbicles have a diameter of about 430 nm, they can pass easily through the pores of most protective masks to reach the sensitive tissues of the patient. The proteins react to the monoclonal antibodies (J1BO1 and J1BO7) and localize on the Golgi body, sexine, nexine and orbicles (but not between the nexine and intine layers), and on the generative cell wall. In the young pollen grain, numerous allergenic protein particles contained in the orbicles and sexine layer, but there is only a small amount of the protein between the nexine and intine layers, since the intine layer is not yet complete at this stage. More will be accumulated there during developmental maturation. The allergenic protein is also found on the tapetal materials remaining in the young anther. Since the materials forming the exine layer and orbicles come from tapetal tissue, it is assumed that some of the allergenic protein is produced in the tapetum and localized in the orbicles and pollen wall during maturation, and that the rest of the allergenic protein is produced in the Golgi body in the mature pollen grain.

Immunolocalization of the cell wall components inPinus densiflora pollen

SummaryIn order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.

Aerodynamic size distribution of the particles emitted from the flowers of allergologically important plants

Abstract The existence of small particles emitted from the dehiscing anther at the time of pollen shedding is shown. We examined pollen samples of five plants causing pollinosis in Japan: Cryptomeria japonica, Alnus japonica, Quercus serrata, Dactylis glomerata and Ambrosia elatior. The pollen samples of C. japonica collected by hydroponic culture had two kinds of particles. The size of these particles was 0.46 to 1.4 μm and 29 to 40 μm (pollen grains) in diameter. The number of the small particles was about 8-fold greater than that of intact pollen grains. Alnus japonica, Quercus serrata and Dactylis glomerata also discharge small particles other than pollen grains although the number is not so large as Cryptomeria japonica. We could not recognize any small particles in a pollen sample of Ambrosia elatior.

Immunoreactive atrial natriuretic peptide in the fish heart and blood plasma examined by electron microscopy, immunohistochemistry and radioimmunoassay

SummaryThe immunoreactivity of atrial natriuretic peptide and ultrastructure of cardiocytes were examined in 5 species each of freshwater and seawater teleosts, as well as in 2 species each of elasmobranchs and cyclostomes. Immunoreactivity was strong in the atria of Cyprinus carpio, Anguilla japonica and Conger myriaster, rather weak in atria of Channa maculata, Lepomis macrochirus, Salmo gairdneri, Oplegnathus fasciatus and Eptatretus burgeri, very weak in atria of Pagrus major, Trachurus japonicus and Triakis scyllia, and not detectable in atria of Hexagrammos otakii, Narke japonica and Lampetra japonica. The immunoreactivity of the atrial cardiocytes was generally stronger in freshwater than seawater fish. Ventricular immunoreactivity was detected only in 7 species, always being weaker than that observed in the atrium. Ultrastructurally, however, secretory granules were found in atria and ventricles of all species examined, being more frequent in the former than the latter. By radioimmunoassay, immunoreactive ANP was detected in the extracts of blood plasma and both atrial and ventricular tissues of all species examined. There were no statistically significant differences in the values between freshwater and seawater species.

Immunochemical and cytochemical detection of wall components of germinated pollen of gymnosperms

To elucidate wall construction of gymnosperm pollen, we studied the distributions of cell wall constituents in the germinated pollen of 14 species of gymnosperms in 8 genera and 6 families cytochemically with 4 staining dyes for polysaccharides, and immunochemically with 4 monoclonal antibodies for polysaccharides and 2 monoclonal antibodies for arabinogalactan proteins (AGPs). All intines and tube walls of the pollen examined contained AGPs and cellulose. Although having small differences, pollen of the Podocarpaceae, Pinaceae, Taxodiaceae, and Cupressaceae (Coniferales) had a similar composition of wall constituents: much AGPs occurred in both the intine and the tube wall, pectins were rare in the tube wall. On the other hand, in Cycas revoluta pollen, pectins were abundant and in Ginkgo biloba , g -(1,3)(1,4)-glucan was abundant in both the intine and the tube wall. The occurrence of g -1, 3-glucan in the intines was common to six Pinus species, but its occurrence in the tube wall differed among them. None of the gymnosperm pollen tubes examined had tube walls similar to those of angiosperm pollen, which consist of two layers, a pectic outer layer and a callosic inner layer. These results suggest that wall formation in gymnosperm pollen tubes is remarkably different from that of angiosperm pollen tubes and the composition of the wall constitutes may reflect a taxonomic relationship among gymnosperms.

Immunolocalization and possible roles of pectins during pollen growth and callose plug formation in angiosperms.

To elucidate the possible roles of pectins during the growth of angiosperm pollen, we studied the distribution and changes in the properties of pectin in the pollen grains and tubes of Camellia japonica, Lilium longiflorum, and five other species at different growth stages by immunoelectron microscopy with monoclonal antibodies JIM5, against de-esterified pectin, and JIM7, against esterified pectin. We also studied the localization of arabinogalactan proteins, which are regarded as pectin-binding proteins, with monoclonal antibodies JIM13 and LM2, against arabinogalactan proteins. Similar results were obtained for all species: JIM5 labeled the intine and part of the callose layer in germinated pollen grains, and labeled the outer layer of the tube wall, the Golgi vesicles, and the callose plug in the pollen germinated in vitro, but did not label any part of immature pollen grains. In contrast, JIM7 labeled the intine of both immature and mature pollen grains, labeled the Golgi vesicles around the Golgi bodies, and strongly labeled the outer layer of the cell wall and the Golgi vesicles in the tube tip region. On the other hand, the distribution of arabinogalactan proteins detected with JIM13 was different for each species, and does not suggest a close relationship between pectin and arabinogalactan proteins. LM2 scarcely reacted with the specimens. We discuss the contribution of pectins to tube wall formation and fertilization and deduce a mechanism of callose plug formation.

Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen ofCamellia japonica

As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically. The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose, arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall.

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

SummaryProtoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

SummaryA polyclonal antibody against β-1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.