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)     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Effects of different light intensities during the forenoon on the afternoon thermal sensation in mild cold

1. 1. The study aimed at knowing whether thermal sensation during afternoon cool exposure could be influenced by bright light (4000 lx) or dim light (200 lx) in the forenoon.

2. 2. The subjects felt cooler after exposure to dim light than to bright light.

3. 3. Melatonin in the urine was significantly higher in bright light than in dim light at 10:30 h and at noon.

     

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.