Purification and cDNA cloning of mouse BM89 antigen shows that it is identical with the synaptic vesicle protein synaptophysin.

The BM89 antigen, first identified in porcine brain by means of a monoclonal antibody, is a neuron-specific molecule widely distributed in the mammalian central and peripheral nervous system (Merkouri and Matsas: Neuroscience 50:53-68, 1992). Here we describe the purification of BM89 antigen from porcine and mouse brain by immunoaffinity chromatography using, respectively, the previously described BM89 monoclonal antibody which belongs to the IgM class and a specific polyclonal antibody generated in the present study. This antibody was also used for the cDNA cloning of the BM89 antigen from mouse brain. cDNA sequencing revealed that the mouse BM89 antigen is identical with the synaptic vesicle protein synaptophysin which is implicated in the control of regulated exocytosis and neurotransmitter release. Mouse BM89 antigen/synaptophysin exhibits, except for one extra amino acid, 100% identity with rat synaptophysin and substantial sequence identity with bovine (92.5% identity) and human (94.8% identity) synaptophysin, but only 59.8% identity with Torpedo synaptophysin. Northern and Western blot analyses confirmed that the mouse BM89 antigen/synaptophysin is expressed only in neural tissues.

Breast cancer imaging with radiolabelled peptide from complementarity-determining region of antitumour antibody.

Specific tumour imaging with radiolabelled monoclonal antibodies has been extensively investigated. Although some success has been reported, there are many limitations due to the slow kinetics, poor extravasation, catabolism by the reticuloendothelial system, and non-specific uptake of macromolecules such as antibodies. We have tried to overcome some of the problems associated with monoclonal antibodies while retaining their specificity by using an antibody-derived synthetic peptide. A synthetic pentadecapeptide (alpha M2) derived from the third heavy-chain complementarity-determining region (CDR-3H) of a tumour-associated monoclonal antibody was produced and shown to retain its specificity against the pan-carcinoma cell-surface antigen, polymorphic epithelial mucin, detected by the parent antibody. The peptide was radiolabelled with technetium-99m and injected intravenously to image malignant lesions in 26 women with primary, recurrent, or metastatic breast cancer. Visualisation of breast tumours and their metastases was obtained shortly after administration of alpha M2, and was optimum by 3 h. Overall, 57 (77%) of 74 sites were visualised. Successful imaging was achieved in 14 of 15 primary tumour sites and all of eight local recurrences. Five of six metastases in the opposite breast, eight of 15 metastatic axillary lymph nodes, and all of six metastatic supraclavicular lymph nodes were imaged. Metastatic sites in the lungs, mediastinum, chest wall, and liver were poorly visualised because of background cardiac blood pool. alpha M2 detected small lesions ( < 2 cm) as efficiently as larger ones. The peptide was rapidly (3 h) cleared from the circulation. No acute or chronic adverse reactions due to the alpha M2 were observed. Specific tumour targeting with the radiolabelled anticancer peptide alpha M2 offers new opportunities for breast cancer imaging and possibly therapy.

Characterization and localization of the BM88 antigen in the developing and adult rat brain.

Monoclonal antibody BM88 identifies a neuron-specific antigen (BM88 antigen) present in the central and peripheral nervous system of the pig (Patsavoudi et al.: Neuroscience 30:463-478, 1989; J Neurochem 56:782-788, 1991). We have previously shown that the antigen is also expressed by cultured neurons derived from newborn rat brain. In the present study we have used the monoclonal antibody BM88 and a specific polyclonal antibody in order to identify the nature of the cross-reactive antigen in rat brain and to investigate its expression and cellular localization in the developing and adult rat nervous system. Western blot analysis and immunocytochemistry revealed that the rat BM88 antigen displays very similar biochemical properties with its porcine homologue. It is a neuron-specific integral membrane protein, apparently not glycosylated, consisting of two 23 kD polypeptide chains. Immunoperoxidase staining demonstrated that the BM88 antigen is widely distributed in the brain of 19-day-old rat embryos. At this stage, immunoreactivity was particularly prominent in differentiated cellular areas and developing fiber tracts of the embryonic rat brain, but was also present in the neuroepithelium. A similar wide distribution of the BM88 antigen was observed in the adult rat brain. Here, immunoreactivity was detected in the neuropil and neuronal perikarya. Immunocytochemical analysis of the expression of the BM88 antigen during postnatal development of the cerebellar cortex showed that this molecule is particularly concentrated in the Purkinje cells between postnatal days 10 to 15; their somata and developing dendrites were distinctly immunopositive during this period. An age-dependent increase in the expression of the BM88 antigen both in brain and in the cerebellum was noted. Electron microscopy confirmed the presence of the BM88 reaction product within the perikarya, axons and dendrites of labeled neurons in the adult brain. The BM88 reaction product was preferentially associated with the limiting membrane of mitochondria, endoplasmic reticulum and small electron-lucent vesicles, but was also present in the plasma membrane, especially at the level of synaptic densities. Our results show that the BM88 antigen participates in an activity common to all or most neurons, and demonstrate that the expression of this antigen is elevated upon neuronal differentiation and maturation.

Monoclonal antibody BM89 recognizes a novel cell surface glycoprotein of the L2/HNK-1 family in the developing mammalian nervous system.

A monoclonal antibody, BM89, obtained with Triton X-114-treated pig synaptic membranes as an immunogen, recognizes a neuronal antigen in the newborn porcine nervous system. By immunohistochemistry, BM89 staining was observed within the neuropil of all areas of the forebrain and spinal cord tested. In addition, BM89 labeled the cell bodies and proximal dendrites of spinal cord neurons. In the peripheral nervous system, BM89 immunoreactivity was present in a subpopulation of dorsal root ganglion neurons and was predominantly associated with non-myelinated axons in peripheral nerves. Initial biochemical characterization of the antigen in pig brain showed that it is an integral membrane glycoprotein with a molecular weight of 41,000. Moreover, it cross-reacts with the L2/HNK-1 carbohydrate epitope expressed by members of a large family of glycoproteins. Homologous antigens with molecular weights of 41,000-43,000 were identified in the rat, rabbit and fetal human brain. Immunoblotting and immunohistochemistry revealed that the epitope recognized by BM89 is developmentally regulated in the rat nervous system. In cryostat sections from rat cerebellum, spinal cord and dorsal root ganglia, an age-dependent decline of BM89 immunoreactivity was observed during postnatal development. In the cerebellum, the BM89 epitope was very abundant in cells of the external and the internal granular layers between postnatal days 5 and 15. During this period some staining was also identified in the developing molecular layer and the prospective white matter. Subsequently, and in the adult, overall staining was greatly reduced and remaining immunoreactivity was associated only with the internal granular layer. In the spinal cord and dorsal root ganglia, staining was very prominent at postnatal day 5; it decreased considerably thereafter and was barely detectable in the adult. Immunostaining of rat brain and dorsal root ganglion cultures revealed that the BM89 antigen is a cell surface molecule expressed by a subpopulation of central and peripheral nervous system neurons. The biochemical properties in conjunction with the topographical location and the developmental profile of the antigen recognized by BM89 suggest that it may represent a developmentally important recognition molecule.