Fumiaki Nishiyama

The staining of golgi membranes with Ricinus communis agglutinin-horseradish peroxidase conjugate in mice tissue cells☆

Ricinus communis agglutinin (RCA)-binding sites were visualized by transmission electron microscopy (TEM) in mice kidney, epididymis and other tissue cells, using the horseradish peroxidase (HRP) as a marker. RCA, a galactose-binding lectin, was conjugated covalently with HRP by the periodate method. Positive reaction with RCA was found at the plasma membrane, Golgi apparatus and multivesicular bodies. Control experiments (e.g. omission of the conjugate, addition of lactose to the incubating medium) established the specificity of the reaction. Plasma membrane staining is in agreement with previously published studies. The staining of the Golgi apparatus was for the first time demonstrated in this study. The positive sites were the cisternal surface of the vesicles and of the saccules of the Golgi apparatus. RCA-HRP staining can be used as a new Golgi marker in cytochemistry.

Changes of lectin-binding sites on the embryonic muscle cell surface in the developing ascidian, Halocynthia aurantium☆☆☆

Abstract Lectin-binding sites on the muscle cell surface of an ascidian embryo were studied using the ferritin labeling technique. The embryos at 4-cell, gastrula, late tail-bud, and larval stages were dissociated in the Ca2+- and Mg2+-free solution with or without collagenase. Dissociated cells and fragments were prefixed, reacted with ferritin-lectin conjugates and processed for electron microscopy. Lectins used were concanavalin A (ConA) and Ricinus communis agglutinin. Ferritin particles showing lectin-binding sites were found singly or in the form of clusters on the cell surface exposed directly to the conjugates. Most of the particles of both conjugates were distributed singly and sparsely on entire surface areas of the 4-cell stage cells, whereas they were rich in population and tended to form clusters when embryos reached the gastrula stage. At the succeeding stages tested, tagged ferritin, which was single or clustering particles, was less in number as compared with those at the former stage; on the surface facing neighboring muscle cells, in particular, the ferritin particles were much fewer than those in areas of notochordal and epithelial sides. It is suggested that the embryonic muscle cells of the ascidian show stage-specific changes of cell surface carbohydrates. They have high reactivity to both lectins around the gastrula stage and bring out the regional difference of both lectin-binding sites in the tail-bud stage, namely during the period of histogenesis.

Concanavalin A binding sites on the erythrocytes of normal and genetically dystrophic chickens

Red blood cells (RBCs) were obtained from genetically dystrophic chickens (Dy) and age-matched controls (C). Dy-RBCs had a lower titer of agglutination to concanavalin A (Con A) compared to C-RBCs. In order to ascertain the difference in agglutination, Con A binding on RBCs was studied, using 125I-labeled Con A ([125I]Con A) and ferritin conjugate to Con A (Fer-Con A). Kinetic analysis of [125I]Con A binding to Dy-RBCs showed a reduction of major binding sites of Con A. There was no difference in the apparent association constant for the major binding sites of Con A between Dy-RBCs and C-RBCs. Quantitative analysis of Con A binding site distribution on RBCs using Fer-Con A showed a remarkable diminution of ferritin particles tagged on the surface of Dy-RBCs. There was no significant difference in the distribution pattern of ferritin particles between Dy-RBCs and C-RBCs.

Localization of concanavalin A binding sites in human pituitary adenoma cells as revealed by HRP-labelling method

SummaryIntracellular lectin (Con A)-binding sites of human pituitary adenoma were examined by electron microscopy using the horseradish-peroxidase (HRP)-labelling technique.In this study were used 16 cases of human pituitary adenomas operated on in our clinics between 1977 and 1981: they concluded 4 each of PRL-, GH-, ACTH-producing and hormonal non-functioning adenomas. In parallel with the detection of lectin-binding sites, basal levels of their secreting hormones were determined by the radioimmunoassay technique, and their producing hormones were characterized light microscopically by the immunocytochemical HRP-labelling technique.In the present study, for hormonal functioning adenoma cells, mature or large granules of each specific type of adenoma cells had no definite Con A-binding sites. On the other hand, immature or small secretory granules of RPL- or GH-producing adenoma cells showed a positive reaction with Con A.ACTH-producing adenomas so far examined revealed no definite binding sites.Some variable results were obtained concerning non-functioning adenomas.Lectins have been and will be very useful in the detection of different subtypes of adenomas in each specific group.

Immunoelectron microscopic analysis of the distribution of tyrosine kinase receptor B in olfactory axons

To determine the morphological basis for the neurotrophic effects of brain-derived neurotrophic factor (BDNF) in the primary olfactory pathway (POP), tyrosine kinase receptor B (TrkB), a membrane-bound receptor for BDNF, was identified and localized in axons of olfactory receptor cells (ORC) of neonatal rat olfactory mucosa using immuno-histochemical and-cytochemical techniques. Initially, the immunospecificity of an anti-TrkB antibody that had been used as a specific antibody for full-length TrkB was confirmed in the olfactory mucosa. Then, a combination of a reduced osmium-LR-White and post-embedding immunogold technique was applied to ORC axons in the lamina propria just beneath the olfactory epithelium. Immunogold particles, which indicate TrkB immunoreactivity, were noted either in close association with the plasma membranes of ORC axons, and designated plasma-lemmal (PL), or within their cytoplasm, and designated cytoplasmic (CP). Most PL particles were seen in the CP portion of the axonal plasma membranes, suggesting that the anti-TrkB antibody binds to the membrane-inserted TrkB that acts as a functional receptor. Some CP particles were on vesicular structures. Quantitative analysis demonstrated that the ratio of CP to PL particles was 7∶3, and this ratio was constant between animals examined (n=5). Because membrane proteins are wrapped in vesicles and transported within the axonal cytoplasm and inserted into the plasma membrane to function there, the present study suggests that TrkB is transported within the cytoplasm of ORC axons and is positioned as a functional receptor for BDNF in their membranes.

Difference of lectin binding sites of secretory granules between normal pituitary and adenoma cells

SummaryElectron-immunocytochemical staining with lectin (concanavalin A: Con A) binding sites analysis was applied to study secretory granules of human pituitary adenomas and surrounding normal pituitary tissue using post-embedded serial ultrathin sections. Twelve cases of human pituitary adenoma and three specimens of normal pituitary tissue surrounding adenomas were studied: the cases were operated on between 1982 and 1984. The tumors consisted of four prolactin (PRL)-, six growth hormone (GH)-, and two adrenocorticotropic hormone (ACTH)-producing adenomas.In parallel with the detection of Con A binding sites of secretory granules, their secreting hormones were characterized electron-microscopically with the immunocytochemical horseradish peroxidase (HRP) labeling using the avidin-biotin technique. The two cases of ACTH-producing adenomas showed either weak or negative reactions with Con A on secretory granules, while normal ACTH-producing pituitary cells showed strong reactions with Con A on every secretory granule observed. Large secretory granules of PRL- or GH-producing cells showed negative reactions with Con A both in the pituitary adenoma and normal pituitary, while some small granulated or sparsely granulated adenoma cells also showed strong reactions with Con A.The complexity of human pituitary adenomas is illustrated as well as the difference in biochemical structure of normal pituitary cells and pituitary adenoma cells secreting the same specific hormone.

Differences in glycoconjugates of adrenocorticotropic hormone-secretory granules between nonadenomatous pituitary cells and adenoma cells as detected by double labeling

Lectin binding sites of adrenocorticotropic hormone (ACTH) secretory granules of human pituitary adenomas and of nonadenomatous pituitary tissue adjacent to adenomas were studied by postembedding immunocytochemical doublestaining on ultrathin sections followed by electron microscopy. The specific hormones produced by the secretory granules were identified by labeling one side of the section with anti-human pituitary hormone antibodies conjugated to gold particles. Simultaneously, the other side was labeled with horseradish peroxidase-lectin to reveal lectin binding sites. Specimens were obtained from four human ACTH-producing pituitary adenomas and from nonadenomatous pituitary tissue surrounding three other adenomas. The four ACTH-producing adenomas showed either weak or negative reactions with concanavalin A, whereas the nonadenomatous ACTH-producing pituitary cells reacted strongly with concanavalin A. Moreover, ACTH secretory granules were significantly larger in the nonadenomatous cells than in adenoma cells. Differences in biochemical structure and ultrastructure between nonadenomatous (normal) pituitary cells and adenoma cells secreting the same specific hormones were demonstrated, and the clinical implications of the results were discussed.

Phosphatase activities in human glioma cells as reaveled by light and electron microscopy — A preliminary study

SummaryAlkaline phosphatase (ALPase) and Mg2+-activated ATPase (Mg2+-ATPase) activities were demonstrated in human brain tumors by light and electron microscopy. Four cases of glioma, i.e., two cases of astrocytoma, grade II, and two cases of glioblastoma, were used as materials.At the light microscopic level, Mg2+-ATPase activity was observed in the capillary wall and glial cells of both astrocytoma and glioblastoma. ALPase activity was restricted to the capillary wall. Its activity was stronger in glioblastoma than in astrocytoma. By electron microscopy, in astrocytoma, reaction product representing Mg2+-ATPase activity was distributed in the plasma membranes of endothelial cells and pericytes. Activity was primarily localized at the abluminal surface of endothelial cells and the surface of pericytes facing endothelium. The plasma membrane of glial cells was also positive. ALPase activity revealed essentially the same distribution pattern in blood vessels as above. In glioblastoma, on the other hand, activities of both phosphatases were markedly positive on the luminal surface of the plasma membrane of endothelial cells. They were much stronger than those along the abluminal endothelial surface.Phosphatase activities in brain tumor appear to change in localization pattern in association with glioma malignancy. This might reflect a functional aspect of changes in blood-brain barrier in glioma.

Intracellular binding of cationized ferritin prolongs the time course of sodium channel inactivation in squid giant axons

SummaryCationized ferritin (CF) applied intracellularly in squid giant axons bound to the negatively charged sites on the cytoplasmic surface of the axolemma. Under the electron microscope, the distribution of CF was found to be dense and uniform over the axolemmal surface. However, the effect of CF on the membrane excitability was highly specific, the major effect being a prolonging of the inactivation time course of the sodium channel without altering the properties of the potassium channel. The binding of CF did not alter the surface potential related to the membrane excitability. When CF was present intracellularly, the time course of the inactivation was characterized by two time constants (slow and fast). The slow component increased with an increase in CF binding and its time constant had a unique value (26 msec) irrespective of the duration of perfusion and concentration of CF. The concentration of CF at which the half-maximum response occurred was about 150nm. Poly-l-glutamate, charged negatively at neutral pH, removed CF from the axolemma and counteracted the CF effect on the sodium channel, although this poly-acidper se did not affect the membrane excitability. Our results indicate that CF binds electrostatically to the inactivation site of the sodium channel but does not affect the voltage sensor, which is supposed to be located deep in the channel.

Morphological Analysis for Neuron‐Like Cells in the Vomeronasal Organ of Human Fetuses at the Middle of Gestation

The vomeronasal organ (VNO) of 5‐month‐old fetuses was examined immunohistochemically by the use of an antiserum to protein gene product 9.5 (PGP). The purpose was to identify if the human fetal VNO is lined by neuroepithelium. The PGP antiserum labeled abundant cells within the vomeronasal epithelium (VE), nerve fiber bundles in its lamina propria, and cells associated with these bundles. PGP‐immunoreactive (ir) vomeronasal epithelial cells were classified into three subtypes. Type I cells, about 44% of the total cells observed, did not have any processes and tended to be located in the basal layer of the VE. Type II cells, about 37% had a single apical process that projected toward the lumen, ending at the epithelial surface. Type III cells sent a prominent process mainly toward the basement membrane, and occupied about 19% of the total cells observed. In the lamina propria, a considerable number of PGP‐ir cells was observed. Some of them were present in nerve fiber bundles and contained processes parallel to the bundles. In addition, PGP‐ir nerve fiber bundles and cells associated with them were even present in the portion of the nasal septal mucosa that was very close to the brain. The present results strongly suggested that the VE in human fetuses at mid‐gestation is a neuroepithelium and that the VE may produce migrating cells toward the brain. Anat Rec, 299:88–97, 2016.