Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins

Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.     

Radioimmunoassay of chenodeoxycholic acid-3-sulfate in urine

A sensitive and specific radioimmunoassay (RIA) for the measurement of urinary total chenodeoxycholic acid-3-sulfate (SCDCA) was developed and the accuracy was confirmed. SCDCA bound to bovine serum albumin as the antigen and emulsified with Freund's complete adjuvant was injected into rabbits. The antiserum obtained was capable of binding 75% of [11,12-3H]SCDCA at 1:1000 dilution. The percentage of bound radioactivity decreased linearly with logarithmic increases in unlabeled SCDCA, from 8 to 200 pmol/ml. The antiserum showed an extremely high specificity for SCDCA (free and conjugated), and the values determined by RIA indicated a close correlation with those found by gas-liquid chromatography. The daily urinary SCDCA level was determined using SCDCA-RIA in 12 disease-free humans and 74 patients with chronic liver diseases. In the normal subjects the daily urinary SCDCA level was 0.74 +/- 0.83 mg/day and increased levels were evident in all groups with chronic liver diseases. The daily urinary SCDCA level corresponds closely with the extent of hepatic dysfunction.     

Induction and Secretion of Pathogenesis-Related Proteins by Salicylate or Plant Hormones in Tobacco Suspension Cultures

Pathogenesis-related proteins (PR proteins), that are inducedin tobacco leaves in hypersensitive response to infection withtobacco mosaic virus (TMV) or by treatment with chemicals, werefound to be also inducible in a dedifferentiated system, tobaccosuspension culture. Quantitative determination of these proteinsusing anti PR 1a IgG showed that their increase started at aboutthe end of cell growth period and that their production couldbe enhanced by the addition of potassium salicylate, Eosin Yellowishand plant hormones (GA3, IAA and 2,4-D). The production dependedon the concentration of the chemical inducer and the cell lineused. In BY-2 cell line, PR proteins amounted to 12 µgat day 5 and then increased exponentially with time, reaching280 µg or 70 µg per g fr wt of cells at day 9 withor without the addition of 25 µM potassium salicylate.More than 90% of the induced PR proteins was found in the mediumand less than 10% in the cells at day 9. Peroxidase activityin the medium was constant throughout the experiment althoughtotal activity in the flask increased with cell growth, indicatingthat PR proteins are actively secreted into the medium. (Received November 12, 1986; Accepted March 6, 1987)     

Purification and partial characterization of membrane-homing receptors in two cloned murine hemopoietic progenitor cell lines

Selective seeding of bone marrow by intravenously transplanted hemopoietic cells depends on the homing receptors of these cells. The receptors are membrane lectins with galactosyl and mannosyl specificities. To purify these lectins, cell membrane was fractionated from two cloned multipotential (B6STU) and bipotential (FDCP-1) hemopoietic progenitor cells. The membrane was solubilized and its proteins were labeled with 125I. The proteins were subjected to affinity column chromatography using galactosyl and mannosyl groups linked to agarose beads. Elution with D-galactose or D-mannose led to specific elution of a single sharp radioactive peak which constituted a constant fraction of membrane proteins. This peak was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by disuccinimidyl suberate cross-linking technique and appeared to have a Mr of 110,000. Under reducing conditions, it consisted of two components with a Mr of 87,000 and 23,000. Treatment with endoglycosidase F indicated about 5% carbohydrate content. Purification of these homing receptors has opened an avenue for the development of immunologic and molecular biologic probes that may help further elucidate the mechanism of homing regulation.     

Differential effect of ik3-1/cables on p53- and p73-induced cell death.

ik3-1/Cables is associated with cdk3 in self-replicating cells. In postmitotic neurons, it may serve as an adaptor molecule, functionally connecting c-abl and cdk5, and supporting neurite growth. Here we report that ik3-1 binds to p53 and p73 in vivo. Ectopically expressed ik3-1 potentiates p53-induced cell death but not p73-induced cell death in U2OS cells. On the contrary, coexpression of ik3-1-DeltaC, an ik3-1 deletion mutant lacking the C-terminal 139 [corrected] amino acids (corresponding to the cyclin box-homologous region), inhibits p73-induced cell death but not p53-induced cell death. ik3-1-DeltaC-mediated inhibition of p73-induced cell death are partially attenuated by overexpression of ik3-1. These data indicate that ik3-1 is not only a regulator for p53-induced cell death but also an essential regulator for p73-induced cell death, and ik3-1-DeltaC competes with ik3-1 only in p73-induced cell death. Furthermore, functional domains of p53 responsible for its interaction with ik3-1 are partially different from those of p73. In conclusion, we found that ik3-1, a putative component of cell cycle regulation, is functionally connected with p53 and p73, but in distinct fashions.     

Chemical analysis of late stages of pheomelanogenesis: conversion of dihydrobenzothiazine to a benzothiazole structure

Pheomelanogenesis is a complex pathway that starts with the oxidation of tyrosine (or DOPA, 3,4‐dihydroxyphenylalanine) by tyrosinase in the presence of cysteine, which results in the production of 5‐S‐cysteinyldopa and its isomers. Beyond that step, relatively little has been clarified except for a possible intermediate produced, dihydro‐1,4‐benzothiazine‐3‐carboxylic acid (DHBTCA). We therefore carried out a detailed study on the course of pheomelanogenesis using DOPA and cysteine and the physiological enzyme tyrosinase. To elucidate the later stages of pheomelanogenesis, chemical degradative methods of reductive hydrolysis with hydroiodic acid and alkaline peroxide oxidation were applied. The results show that: (1) DHBTCA accumulates after the disappearance of the cysteinyldopa isomers, (2) DHBTCA is then oxidized by a redox exchange with dopaquinone to form ortho‐quinonimine, which leads to the production of pheomelanin with a benzothiazine moiety, and (3) the benzothiazine moiety gradually degrades to form a benzothiazole moiety. This latter process is consistent with the much higher ratio of benzothiazole‐derived units in human red hair than in mouse yellow hair. These findings may be relevant to the (photo)toxic effects of pheomelanin.     

Magnesium ion: a possible physiological regulator of aldosterone production

We examined the direct effect of magnesium ion on aldosterone production by adrenal cells using collagenase-dispersed zona-glomerulosa cells in rats. The effects of magnesium on aldosterone production stimulated by angiotensin II or ACTH were also investigated. Both magnesium sulphate (MgSO4) and magnesium chloride (MgCl2) (0 to 2 mM) decreased aldosterone production in a dose-dependent manner. In comparison with magnesium-free medium, 2 mM MgSO4 inhibited aldosterone production by 73% and MgCl2 by 65%. In addition, MgSO4 showed an inhibitory effect on aldosterone production stimulated by angiotensin II (10pM to 10nM), whereas it had no significant effect on aldosterone production due to ACTH stimulation (10pM to 10nM). These data suggest that magnesium has an inhibitory action on aldosterone production in vitro and may be a physiological regulator of aldosterone production.     

Effect of seaweed preparations on murine immunocytes

During a screening programme for new medical agents, many aqueous extracts from 59 species of seaweed were found to possess bioactivity against murine immunocytes. Thirty-eight extracts (8 green, 12 brown, 18 red algae) showed suppressive effects on the mitogenic response. Furthermore, 16 extracts (2 green, 6 brown, 8 red algae) suppressed the production of Interleukin 1 (1L-1) from murine macrophage. Using the murine mixed lymphocyte reaction assay, suppressive effects were observed in 4 red algae, but none in green or brown algae. Nine seaweed extracts suppressed the production of secondary antibody (IgG, IgM). Extracts of 3 red algae suppressed strongly the proliferation of bone marrow cells, but 2 other red algae caused stimulation above 200%. This is apparently the first report showing immunosuppressive activity from marine algae.