1. When rat brain or superior cervical ganglion monoamine oxidase was incubated with increasing concentrations of clorgyline, using tyramine as substrate, the inhibition of the enzyme could be represented by a pair of sigmoidal curves joined by a horizontal region where inhibition was constant. Tyramine appeared to be metabolized by two enzymes, one of which was highly sensitive to clorgyline, designated A, whereas the other enzyme, designated B, was less sensitive to clorgyline.2. The ratio of A/B activity for brain was 6/4 while in the ganglion it was 9/1.3. When the experiments were repeated using noradrenaline as the substrate, the inhibition of the enzyme followed a simple sigmoidal curve where deamination was inhibited by low concentrations of clorgyline as observed with enzyme A.4. We conclude that tyramine is deaminated by both A and B enzymes whereas noradrenaline is deaminated only by enzyme A, the enzyme which is most active in the ganglion. Our observations are consistent with the hypothesis that a specific intraneuronal monoamine oxidase plays an important role in the catabolism of noradrenaline in sympathetic nerves.

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Identification and immunolocalization by monoclonal antibody of NSP-5, a surface polypeptide of neural cells.

A monoclonal antibody, termed anti-NSP-5 (anti-Neural cell Surface Protein-5) was obtained from an hybridoma generated by fusing rat myeloma cells with splenocytes of a rat immunized with membranes from the cerebella of weaver mutant mice. This antibody reacted with the surface membrane of a subset of neurones in cultures from cerebella and dorsal root ganglia. In both culture systems, only tetanus toxin-positive cells were stained by the antibody. In sections of adult cerebellum a punctate pattern of staining was seen in the molecular layer, the Purkinje cell layer and the upper part of the granule cell layer. The white matter was strongly positive whereas granule cell and Purkinje cell bodies were clearly negative. In sections from adult dorsal root ganglia anti-NSP-5 labeled most sensory neurones including their axones in the dorsal roots. The expression of the antigen was developmentally regulated. It could not be detected in cerebellar cultures prepared from animals younger than 7 days, in good agreement with the data obtained on tissue sections. Similarly, the antigen could not be detected by immunoblotting in neonatal spinal cord, but a NSP-5-reactive band was present at postnatal day 7. The antibody bound a polypeptide of around MW 180 000 in extracts prepared from adult mouse spinal cord or cerebellum. When purified by immunoaffinity chromatography the antigen co-eluted with numerous strongly associated polypeptides. Upon subcellular fractionation most of it remained associated with a Triton-X100 insoluble fraction thus co-distributing with the cytoskeleton.

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Characterization of an antiserum to synaptic glomeruli from rat cerebellum.

Rabbit anti-rat cerebellar synaptic glomeruli antiserum when absorbed with non-neural tissues reacts only with neural tissues when tested by indirect immunofluorescence on tissue sections. Further absorption with forebrain results in the antiserum which detectably reacts only with synaptic glomeruli and soma of Purkinje cells of both rat and mouse. The developmental expression of the synaptic glomeruli antigen(s) parallels the formation of synapses between mossy fibers and granule cells. Immature synaptic contacts do not contain recognizable antigen(s), whereas only at postnatal Day 15 glomeruli become antigen-positive. At this stage antigen in Purkinje cells is no longer carried in their dendrites, but becomes confined to the cell soma. Staggerer mutant mice still express the immature pattern of antigen distribution on postnatal Day 18.

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A biochemical analysis of the secregation of HLA-DR heavy and light chains in a family studied by two-dimensional gel electrophoresis.

Previous studies in our laboratory using primed lymphocyte typing (PLT) in an informative family have led to the identification of four traits and two regions, separable by recombination within the HLA-DR system. In the present study, we analysed the heterogeneity of HLA-DR antigens among members of this family as a first step towards defining the molecular entities responsible for the observed cellular reactivities. Cell lines of various genotypes derived from the members of this family were labelled biosynthetically with 35S-methionine. The total glycoproteins and immunoprecipitates prepared with two monoclonal anti-HLA-DR antibodies, two monoclonal anti-mouse Ia antibodies cross-reactive with HLA-DR, and a rabbit anti-HLA-DR (anti-p27,33) antiserum were fractionated by two-dimensional gel electrophoresis. In agreement with previous results, HLA-DR light chains were clearly polymorphic whereas the heavy chains looked rather monomorphic. The light chain subunits either segregated with one haplotype, or were shared by some but not all haplotypes or were common to all haplotypes. Whereas the haplotype-specific polypeptides correlated best with HLA-DR3 and HLA-DR5 specificities, the shared subunits may have corresponded to the cross-reactivities observed by PLT. Comparison of the patterns obtained with the various monoclonal antibodies and with the rabbit antiserum revealed that each monoclonal antibody bound a subset of HLA-DR light chains, all of which were present in the anti-p27,33 precipitates. This rabbit antiserum and one of the monoclonal antibodies immunoprecipitated a set of heavy chains not bound by the other 3 antibodies. Since overlapping pools of light chains were present in all five immunoprecipitates, these results suggest that different heavy chains may associate with the same light chains.

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Adult and embryonic mouse neural cell adhesion molecules have different binding properties.

Interactions between neural cell surfaces seem to be of prime importance during neuroontogenesis, and responsible for the guidance of migrating neuroblasts and growing axons and for the formation of synapses. Little is known about the underlying molecular mechanisms, but most hypotheses imply the existence of cell-surface molecules that mediate the formation of transient or permanent bonds between neural cells. Recently, a membrane glycoprotein called neural cell adhesion molecule (N-CAM) has been characterized in chick and rodent nervous tissue that appears to act as a ligand in adhesion among neural cell bodies or neurites. We have identified a mouse neural surface glycoprotein, named BSP-2 (ref. 7), which by criteriaof electrophoretic migration, developmental changes, amino acid and sugar composition seems to be closely related or identical to N-CAM. Both BSP-2 (refs 8, 9) and N-CAM undergo conversion from an embryonic to an adult form during brain development and it has been suggested that this transition changes the adhesive properties or the binding specificity of the molecule. Using a neuroblastoma line to study functional differences between embryonic and adult BSP-2/N-CAM molecules, we show here that liposomes bearing adult BSP-2 but not those bearing the embryonic form adhere to neuroblastoma cells, demonstrating that the two forms do indeed possess different binding properties.

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Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1.

Cell surface molecules have been implicated in cell interactions which underlie formation of the nervous system. The analysis of the functional properties of such molecules has profited from the combined use of antibodies and cell culture systems. It has been suggested that the interplay between these molecules modulates cell-to-cell interaction at critical developmental stages. In the mouse, N-CAM and L1 antigen have been shown to mediate Ca2+-independent adhesion among neural cells. N-CAM plays a role in fasciculation of neurites and formation of neuromuscular junction. L1 is apparently not involved in synaptogenesis, but in migration of granule cell neurones in the developing mouse cerebellar cortex. The two antigens are distinct molecular and functional entities which act synergistically in aggregation of neuroblastoma and early postnatal cerebellar cells. In view of a certain similarity in function between the two groups of molecules, it was not surprising to find that structural similarities are detectable by the monoclonal antibody L2. We show here that a carbohydrate moiety recognized by L2 and HNK-1 monoclonal antibodies, is present in mouse N-CAM and L1. The L2 epitope appears on all major neural cell types but not all N-CAM molecules express it. This heterogeneity points to a previously undetected molecular diversity which may have functional implications for modulating cell adhesion during development.

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Glial cells express N-CAM/D2-CAM-like polypeptides in vitro.

The joining together of neurites to form fascicles and the growth of axons along glial surfaces during early development suggest that neurone-neurone and neurone-glial adhesion interactions are of considerable importance for defining nerve tracts. In vitro studies have indicated that adhesion between neurones involves a glycoprotein that has been independently studied under the names of N-CAM (for neural cell adhesion molecule), D2-CAM and BSP-2 (refs 10, 11). As N-CAM/D2-CAM appears to be a homophilic ligand that binds to N-CAM/D2-CAM polypeptide on adjacent cells, this glycoprotein is potentially important in adhesion interactions between any two N-CAM/D2-CAM-expressing cells. While it has been suggested that neurone-glial adhesion involves molecules other than N-CAM/D2-CAM, it is known that N-CAM/D2-CAM antigenic determinants are expressed by glial cells in vivo and that injection of anti-N-CAM antibodies into the eye-cup of chick embryos disrupts normal patterns of neuritic apposition to glial endfeet in the developing optic stalk. Do the molecules expressed by glia share restricted antigenic determinants, or binding domains, with N-CAM/D2-CAM, or are N-CAM/D2-CAM polypeptides expressed by glia? Here we present immunocytochemical evidence which suggests that all classes of macroglia express N-CAM/D2-CAM antigenic determinants on their surfaces and immunochemical analyses which indicate that the molecules expressed by purified astrocytes are closely similar, or identical, to at least some forms of N-CAM/D2-CAM obtained from whole brain or purified neurones. However, our results also suggest that different N-CAM/D2-CAM polypeptides may be separately expressed by neurones and astrocytes.

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