Masatsune Ishiguro

Oligomannose-coated liposomes as an adjuvant for the induction of cell-mediated immunity

The effect of the coating of ovalbumin‐reconstituted liposomes with various oligosaccharides on their immunogenicity was investigated in mice. The coating of liposomes with oligomannose or yeast mannan drastically enhanced their ability to induce an ovalbumin‐specific delayed‐type footpad swelling response with a peak at 24 to 48 h post‐challenge. Among various oligosaccharides tested, only those with mannose residue at the non‐reducing termini manifested the activity when applied to liposomes. Since such oligosaccharides are ubiquitously found in the body, these results suggested the usefulness of oligomannose‐coated liposomes as a safe adjuvant for the induction of cell‐mediated immunity.

Electrophoretic separation of proteins on agarose gel—Application for direct immunization with a gel piece containing an antigen

An efficient method of separating proteins by electrophoresis with agarose gels containing sodium dodecyl sulfate is described. The separated proteins were then used to make antibodies. The separation of proteins in the molecular weight range 25K to 94K on 4 to 5% agarose gels showed good resolution which was comparable to that obtained by electrophoresis with polyacrylamide gels containing sodium dodecyl sulfate. The subunits of human chorionic gonadotropin were separated by the preparative agarose gel electrophoresis and the gel bands containing the subunits were used directly to raise antibodies against these proteins. The degree of solubilization of the proteins from the gel slices was found to be complete.

PATHOLOGICAL STUDY ON MUCOSAL CHANGES IN SMALL INTESTINE OF RAT BY ORAL ADMINISTRATION OF RICIN: I. Microscopical Observation

A histological study on small intestine of Wistar rats after oral administration of ricin, a proteinous toxin from castor bean seeds, was carried out. In Experiment I, the jejunum was examined at 1, 2, 5, 10, 15, 20, 24, and 40 hours after oral administration of ricin 30 mg/kg. In Experiment II, ricin was administered at dose of 1, 3, 5, 10, 15, 30, and 60 mg/kg, and after 5 hours the jejunum, mid‐portion, and ileum were examined. For comparison, ricin 0.5 mg/kg was administered intraperitoneally and castor bean hemagglutinin (CBH) 30 mg/kg orally. In both experiments, the changes of mucosa were essentially similar dependent on time‐lapse and dose respectively, which were atrophy of villus, elongation of crypt, degeneration of epithelium, decrease of goblet cell, fusion of intervillous epithelia, infiltration of neutrophils and eosinophils, and dissociation between epithelium and lamina propria. These changes were most manifest in the jejunum that was contacted with ricin first in a high concentration. In contrast, intraperitoneal administration of ricin caused only dissociation between epithelium and lamina propria and oral administration of CBH caused only milder atrophic changes. The evidences suggest that the mucosal changes by oral administration were caused by direct contact with ricin.

Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.

Intracellular molecular species of human chorionic gonadotropin from normal but non-cultured first trimester placental tissues

Intracellular forms of human chorionic gonadotropin (hCG) were analyzed by SDS-polyacrylamide gel electrophoresis, protein blotting and immunological techniques in normal but non-cultured first trimester placentae. Placental cells were found to contain the major components of the 23K and 19K forms of the beta-subunit and the 21K form of the alpha-subunit of hCG which remained sensitive to endoglycosidase H and Con A-Sepharose 4B and small amounts of mature (urinary) subunits. An unknown molecular species of the alpha-subunit (Mr = 17K) that was not bound to Con A-Sepharose 4B was also detected. These intracellular molecular species accumulated in the placentae mainly during the first trimester. These results suggest that hCG subunits accumulate in placental cells as predominant intermediates containing high-mannose oligosaccharides.

Characterization and comparison of two forms of human chorionic gonadotropin from hydatidiform moles with low and high immunoreactivity

Two glycoproteins (LM-hCG and HM-hCG) with gonadotropic activity were purified from the chorionic tissue of patients with hydatidiform mole with low and high urinary human chorionic gonadotropin levels and were compared to each other. The immunologic, biologic, and physicochemical properties of the two preparations were very similar. Also, the quantities of LM-hCG and HM-hCG recovered from molar tissue from the two types of patients were similar. However, the molar tissue from patients who excreted high levels of human chorionic gonadotropin immunoreactivity also contained other molecular forms with apparent human chorionic gonadotropin immunoreactivity tissues from patients who excreted low levels did not. These latter molecular forms may account for the differences in the levels of human chorionic gonadotropin immunoreactivity excreted by the two types of patients.

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase.

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.

Studies on Immunoreactive ACTH from Human Term Placenta

Extracts of human term placenta were fractionated by Sephadex G-75 gel filtration and assayed for immunoreactive ACTH. Both high and low molecular weight protein fractions were detected to be immunologically reactive toward anti-human ACTH (1--39 alpha) antibody. For the extraction of low molecular weight ACTH from human term placenta (pl. -ACTH), a glacial acetic acid-acetone mixture was employed, while a pH 3.0-HCl solution was used for high molecular weight immunoreactive ACTH. The high molecular weight immunoreactive ACTH fraction (F-I), co-eluted with horse hemoglobin from a Sephadex G-75.column in 0.1M acetic acid, was essentially devoid of low molecular weight materials as revealed by polyacrylamide gel disc electrophoresis at pHs 9.5 and 4.3. Tryptic digestion of F-1 at pH 8.1 and 37 degrees C for 4 hr with E/S of 1/100, followed by fractionation with a Sephadex G-75, resulted in the formation of lower molecular weight fragments. One fragment was eluted at the same position as that of porcine ACTH with a recovery of 86% of immunoreactivity of F-I. Another fragment which was eluted last exhibited positive beta-endorphin receptor binding activity. These results suggest the presence of a common precursor protein to ACTH and beta-endorphin in human term placenta.