Differential spectrum of expression of neural cell adhesion molecule isoforms and L1 adhesion molecules on human neuroectodermal tumors.

A series of four medulloblastomas, seven neuroblastomas (Nb), two ependymomas, and three gliomas, human neuroectodermal tumors, were screened for their expression of adhesion molecules L1, carcinoembryonic antigen, neural cell adhesion molecule isoforms (N-CAM) and HNK1 epitope by Western blotting and double immunofluorescence labeling. All seven neuroblastomas, whatever their differentiated state, expressed L1, a neural cell surface developmental antigen, whereas all other tumors tested were negative. All tumors expressed N-CAM; however, a large diversity was observed among the isoforms. Low sialylated N-CAM 140 was present, with different intensity, in ependymomas and astrocytomas. High sialylated isoforms were detected by a monoclonal antibody (anti-MenB) specifically recognizing high polymers of alpha 2-8 linked neuraminic acid. They were expressed in all medulloblastomas studied (4 of 4), and in 4 of 7 Nbs examined. Negative cases corresponded to tumors having undergone chemotherapeutic treatment or to ganglioneuroma. The interconversion from high to low sialylated forms might reflect changes which are critical for the conversion of Nbs into benign ganglioneuromas. HNK1 epitope was expressed on a large diversity of molecules by nearly all tumors except two Nbs which were also negative with anti-MenB antibody. This simultaneous loss of carbohydrate epitopes could correlate with higher maturation states of the tumors. None of the tumors expressed carcinoembryonic antigen. Therefore, anti-L1 and anti-MenB antibodies define differentiation-related antigens that could differentiate between Nbs and other tumors and may prove helpful in diagnosis and understanding of the biological nature of neuroectodermal tumors. An immunodot assay was set up and allowed to titrate the presence of polysialic acid units in cerebrospinal fluid from patients presenting meningeal spread of medulloblastomas. It could help to assess metastasis and to monitor the effects of chemotherapeutic treatment on polysialylated N-CAM positive tumors.

Antibody against Bacillus thuringiensis phosphatidylinositol-phospholipase C: some examples of its potential uses.

We have purified the phosphatidylinositol-phospholipase C enzyme from the bacterium Bacillus thuringiensis. This enzyme is able to release in soluble form molecules which are anchored to membranes via a glycan-phosphatidylinositol group. It exhibits a molecular weight of 33-35 kDa. We raised polyclonal antisera against the molecule and used them in immunoblot as well as radioimmunoassays for enzyme detection. This last technique should facilitate monitoring of chromatographic steps during enzyme purification. We coupled antibodies to Sepharose beads in order to remove the enzyme from incubation media. This reagent also proved to be particularly useful in control experiments designed to ascertain that the observed release of molecules is due to the action of the phosphatidylinositol-phospholipase C enzyme and not to spontaneous release or to cleavage by nonspecific hydrolases. A search for cross-reactive molecules in other bacterial strains or mammalian tissues gave negative results. This leads to the conclusion that a great diversity exists between phosphatidylinositol-phospholipases C, even among different bacterial strains.

Antipeptide antibodies to the beta 2-adrenergic receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop.

We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung beta 2-adrenergic receptor (amino acids 16-31 and 174-189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified beta 2-adrenergic receptor in immunoblot conditions when used at a dilution of 1:500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as 125I-cyanopindolol labeled beta 2-adrenergic receptor. This last result implies that the recognized epitopes do not contain the 125I-cyanopindolol binding domain of the beta 2-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified beta 2-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native beta 2-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing beta 2-adrenergic receptor-like molecules exhibited a neuronal phenotype.