A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy

Summary A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immuno-histochemical researches.     

Phencyclidine-induced expression of c-Fos-like immunoreactivity in mouse brain regions

For the purpose of studying a role of immediate early genes in psychotomimetic-induced behavioral excitation, we experimentally enhanced the locomotor activity of mice by acute administration of phencyclidine and examined the expression and localization of the c-Fos-like and c-Jun-like immunoreactivities in brain regions. A single injection of phencyclidine (5.0 mg/kg, i.p.) significantly increased not only the locomotor activity but also the expression of c-Fos-like immunoreactivity in several brain regions, particularly in the parietal cortex, hippocampal dentate gyrus, piriform cortex and hypothalamus. Interestingly, the c-Fos-like immunoreactivity in the parietal cortex continued to increase for 1 week after the phencyclidine injection. These results indicate that phencyclidine, even injected only once, can induce the persistent expression of c-Fos or c-Fos-related protein(s) in the mouse brain, and also suggest the possibility that such a c-Fos expression may underlie the behavioral and/or psychotomimetic effects of phencyclidine.     

Absorption of elastase through the jejunal mucosa of the rat

Summary In order to reveal the absorption process of elastase from the intestine, hog pancreatic elastase was injected into the ligated jejunum lumen of the rat, and the tissues were cytochemically observed at various times after injection. The peroxidase anti-peroxidase (PAP) method using anti-hog-elastase rabbit antibody was used for light microscopy, and the anti-elastase Fab-peroxidase conjugate was used for electron microscopy. The tissues stained by the PAP method exhibited a dense deposition of reaction products on the luminal surface of epithelial cells and a moderate deposition in the blood and lymph capillaries of the intestinal villi. Immunoelectron microscopy revealed that the reaction product was deposited on the surface of the microvilli and in their pocketing; some was found in the pinocytotic vesicles in the terminal-web area and on the inner surface of the enlarged smooth endoplasmic reticulum. Round droplets which gave a positive reaction were found in the widened intercellular cleft and the thick basement membrane lining the blood capillaries and lymphatics. The jejunum retained its normal ultrastructure. The results indicate that the elastase molecules, which were introduced into the rat jejunum lumen, were absorbed without being decomposed through healthy intestinal epithelial cells by pinocytosis and translocated into blood and lymph capillaries.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

A novel mannose-specific and sugar specifically aggregatable lectin from the bark of the Japanese pagoda tree (Sophora japonica)

A new D-mannose/D-glucose-specific lectin (B-SJA-II) was isolated from the bark of the Japanese pagoda tree, Sophora japonica. B-SJA-II was separated from a well known D-galactose/N-acetyl-D-galactosamine-specific lectin (B-SJA-I) by affinity chromatography on lactamyl-Sepharose, then purified by affinity chromatography on maltamyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B-SJA-II gave four bands: subunit a-1 (Mr = 19,400), a-2 (Mr = 18,200), b-1 (Mr = 15,000), and b-2 (Mr = 13,200). Carbohydrate analysis and binding study with horseradish peroxidase-labeled lectins on the bands electroblotted onto polyvinylidene difluoride membrane showed that the three subunits other than b-2 have N-linked oligosaccharides typical of plant glycoproteins. The binding assay with horseradish peroxidase-glycoproteins revealed that all the subunits can bind sugar specifically with fetuin and asialofetuin. Furthermore, B-SJA-II aggregated to form precipitates in the absence of a specific sugar and became soluble upon addition of the specific sugar. The results indicate that each subunit has a sugar-binding site for the mannosyl core of N-linked oligosaccharide chains and recognizes each other sugar specifically to form aggregates. According to the N-terminal amino acid sequences obtained, the subunits are classified into two groups. The first group (a-1 and a-2) has an N-terminal sequence 50% identical with that of other S. japonica lectins (Hankins, C. N., Kindinger, J. I., and Shannon, L. M. (1988) Plant Physiol. 86, 67-70) and the amino acid sequence initiating at position 123 of concanavalin A (Cunningham, B. (1975) J. Biol. Chem. 250, 1503-1512), while the N-terminal sequence of the second group (b-1 and b-2) is homologous to that of concanavalin A, but completely different from that of the first group.     

Real-Time Monitoring of Slow-Wave Sleep by Electroencephalogram Variance

Based on statistical variance as an index of electroencephalogram (EEG) parameters, we monitored slow-wave sleep in both humans and rats in real time and on-line with a widely used personal computer. This EEG variance method may be a useful tool to carry out biological rhythm research, including sleep studies.