Chemoreceptors expressed in taste, olfactory and male reproductive tissues.

We have identified three genes encoding previously uncharacterized chemoreceptors expressed in rat sensory and reproductive tissues using a reverse transcriptase polymerase chain reaction strategy. Degenerate oligonucleotides designed from conserved sequences in the rat olfactory receptor gene family were used to amplify candidate receptor gene products expressed in taste tissue. Sequence analysis of three distinct clonal isolates revealed that the gene products from taste bud were 30-75% identical to previously identified olfactory receptor genes. The genomic coding sequences predicted protein structures with seven membrane spanning regions that have strong conservation relative to other members of the G-protein-coupled olfactory receptor gene family. Transcripts for each of the three gene products were detected exclusively in taste, olfactory and male reproductive tissue. Sequence analysis of the polymerase chain reaction products confirmed that identical transcripts were expressed in all three tissues. These findings are the first demonstration that identical olfactory receptor-like gene are expressed in three distinct tissues.

Induction of L1 mRNA in PC12 cells by NGF is modulated by cell-cell contact and does not require the high-affinity NGF receptor.

We examined the effects of nerve growth factor (NGF) and cell-cell contact on expression of the neural cell adhesion molecule L1 in PC12 cells. After 7 d exposure to NGF, but not after exposure to EGF, FGF, TGF beta, or dibutyryl cAMP (dbcAMP), L1 mRNA levels increased fourfold. This increase was not blocked by K252a, an inhibitor of the high-affinity NGF receptor, although neurite extension was completely inhibited. L1 mRNA levels also increased in NGF-treated mutant PC12 cells (PC12nnr5) that lack the high-affinity NGF receptor. The effect of NGF on L1 mRNA was greatest in cells cultured at high density, but its effect on cells cultured at low density was augmented by antibody to L1 (to mimic L1 homophilic binding). Various extracellular matrix components had no differential effects on L1 mRNA levels in either the presence or absence of NGF. Together, these findings suggest that NGF regulates L1 expression by a mechanism that is independent of the high-affinity NGF receptor and that this regulation is modulated by cell-cell contact but not by cell-extracellular matrix interactions.

Expression of the neural cell adhesion molecule (NCAM) and polysialic acid during taste bud degeneration and regeneration.

Taste receptor cells are replaced throughout life, accompanied by continuing synaptogenesis between newly formed taste cells and first-order gustatory fibers. The neural cell adhesion molecule (NCAM) is expressed by a subset of taste cells in adult rodents and appears on gustatory nerve fibers during development prior to differentiation of the taste buds. We employed antibodies against the extracellular domain of the NCAM polypeptide (mAb 3F4) and against polysialic acid (PSA) residues found on embryonic forms of NCAM (mAb 5A5) to investigate the relationship between the expression of these molecules and the innervation of taste buds in adult rats. In unoperated rats, anti-NCAM recognized a subset of cells within the vallate taste buds and also the fibers of the glossopharyngeal (IXth) nerve, including those innervating the gustatory epithelium. Taste bud cells did not express PSA but mAb 5A5 immunoreactivity was observed on some fibers of the IXth nerve, including a few that entered the taste buds. Bilateral crush of the IXth nerve resulted in the loss of NCAM expression from the gustatory epithelium within 8 days. As IXth nerve fibers reinnervated the epithelium, NCAM expression was seen first in the nerve, followed by increased expression in the epithelium as the taste cells differentiated from their precursors. PSA expression by fibers of the IXth nerve did not return to normal until well after the regeneration of the vallate taste buds. The present results demonstrate that taste cell expression of NCAM is dependent upon innervation by the IXth nerve and that NCAM expression appears in the nerve prior to its expression in the differentiating epithelium during regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

VASE exon expression alters NCAM-mediated cell-cell interactions.

The neural cell adhesion molecule (NCAM) is found on cells as several related polypeptides formed by alternative splicing of the single NCAM gene. The alternatively spliced 30-bp VASE exon in the fourth immunoglobulin-like domain is the structural variation nearest those portions of the polypeptide proposed to mediate cell-cell adhesion. To test the ability of distinct forms of the NCAM molecules to mediate cell adhesion, L cells were transfected with expression vectors encoding rat 140 kD NCAM +/- the VASE exon. L cell lines which expressed these polypeptides were isolated and tested for self-aggregation in a low shear, rapid aggregation assay. Increased cellular aggregation of the transfectants was observed to be a function of the NCAM molecule expressed. These transfected cells showed segregation in a long term co-aggregation assay: cells expressing NCAM--VASE formed aggregates which tended to exclude cells expressing NCAM+VASE and vice versa. These results provide direct evidence that this small difference in NCAM structure is sufficient to allow segregation of cells.

Taste bud expression of human blood group antigens.

Some human blood group antigens are expressed by rodent epithelial cells at different stages of differentiation. Since adult taste cells are continually replaced throughout life, we investigated the expression of the H, B, A and Lewisb blood group determinants by cells of the rat fungiform, foliate and vallate papillae. We employed antibodies against the trisaccharide structures of the H, B, and A blood group antigens and against the Lewisb blood group epitope in studies of normal and denervated taste buds. The antibody against the H antigen reacted with the majority of cells in all taste buds and with cells in the spinous layer of the tongue epithelium. The B antigen was expressed by the majority of taste cells but not by other epithelial cells. The expression of the A antigen was significantly less in the fungiform taste buds than in the vallate or foliate taste buds. The A antigen was also abundantly expressed in the acini of the lingual salivary glands. The Lewisb epitope was expressed by a subset of cells in taste buds of the fungiform, foliate and vallate papillae. Taste buds are trophically dependent upon gustatory nerve innervation. Transection of the chorda tympani or the IXth nerve resulted in the loss of expression of these molecules from the gustatory epithelium, indicating that they are expressed only on differentiated taste cells. The blood group antigens are lactoseries carbohydrates; they are differentially expressed in developing cochlear hair cells and olfactory neurons and may play roles in cell-cell recognition, adhesion, and other interactions important in the developing nervous system. They could have similar functions in the taste and olfactory systems, where the receptors are continually renewed and new synapses between the receptors and their neural targets continually form.

NCAM expression by subsets of taste cells is dependent upon innervation.

The expression of the neural cell adhesion molecule (NCAM) and distinct carbohydrate groups by cells of the taste buds of the rat vallate papilla was investigated by immunohistochemical and biochemical techniques. We employed antibodies against 1) the extracellular (mAb 3F4) and cytoplasmic (mAb 5B8) portions of the NCAM polypeptide, 2) the highly sialylated form of NCAM (mAb 5A5), 3) carbohydrate epitopes associated with glycosylated NCAM forms in the rat (mAb 2B8) or frog (mAb 9-OE) olfactory system, and also 4) the Lewisb blood group carbohydrate epitope (mAb CO431). NCAM mRNA was demonstrated by polymerase chain reaction (PCR) in samples of the vallate papilla, suggesting the presence of NCAM in cells of the taste buds. Antibodies against NCAM (mAbs 3F4 and 5B8) recognized a subset (about 20%) of cells within the vallate taste buds; fibers of the glossopharyngeal nerve, including those innervating the gustatory epithelium, were NCAM immunoreactive. Taste bud cells did not express polysialic acid (mAb 5A5), but mAb 5A5 immunoreactivity was observed on fibers of the IXth nerve, including a few that entered the taste buds. All or nearly all of the cells within the vallate taste buds were immunoreactive to mAb 2B8, whereas mAbs 9-OE and CO431 reacted with subsets of cells. The carbohydrates recognized by mAbs 2B8 and 9-OE were also abundantly expressed in the ducts and acini of the lingual salivary glands. Bilateral crush of the IXth nerve resulted in the loss of expression of all of these molecules from the gustatory epithelium. If cells of the taste bud express NCAM during their final stage(s) of differentiation, then NCAM could play a role(s) in the growth of gustatory axons toward their target epithelial cells and in the recognition between the nerve fibers and mature taste receptor cells, or among the taste bud cells themselves.

Distinct subsets of sensory olfactory neurons in mouse: possible role in the formation of the mosaic olfactory projection.

The axons of the primary sensory olfactory neurons project from the olfactory neuroepithelium lining the nasal cavity, onto glomeruli covering the surface of the olfactory bulb. Neuroanatomical studies have shown previously that individual olfactory glomeruli are innervated by neurons that are dispersed widely within the nasal cavity. The aim of the present study was to test the hypothesis that phenotypically unique subsets of primary sensory olfactory neurons, scattered throughout the nasal cavity, project to a subset of glomeruli in specific olfactory bulb loci. Immunochemical and histochemical analyses in neonatal mice revealed that the plant lectin, Dolichos biflorus agglutinin, bound to a subset of mature primary sensory olfactory neurons which express the olfactory marker protein. This subset of neurons was principally located in the rostromedial and dorsal portions of the nasal cavity and projected specifically to a subset of glomeruli in the rostromedial and caudodorsal portions of the olfactory bulb. Analysis of Dolichos biflorus-reactive axons revealed that these axons coursed randomly, with no evidence of their selective fasciculation, within the olfactory nerve. It was only at the level of the rostral olfactory bulb that a significant reorganisation of their trajectory was observed. Within the outer fibre layer of the bulb, discrete bundles of lectin-reactive axons began to coalesce selectively into fascicles which preferentially oriented toward the medial side of the olfactory bulb. These data demonstrated that a phenotypically distinct subset of primary sensory olfactory neurons exhibits a topographical projection from the olfactory epithelium to the olfactory bulb, and suggests that these, and other subsets, may form the basis of the mosaic nature of this pathway. Moreover, it appears that the outer nerve fibre layer in the rostral olfactory bulb plays an important instructive role in the guidance and fasciculation of olfactory sensory axons.

Axon growth is enhanced by NCAM lacking the VASE exon when expressed in either the growth substrate or the growing axon.

The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM +/- VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM - VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM +/- VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM - VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM +/- VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM - VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM - VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a significant factor in the reduced axonation capabilities of mature neurons.

The neural cell adhesion molecule (NCAM) heparin binding domain binds to cell surface heparan sulfate proteoglycans.

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using beta-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10(-7) M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell-HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites.

Delineation of olfactory pathways in the frog nervous system by unique glycoconjugates and N-CAM glycoforms.

The olfactory neuroepithelium, which contains the primary sensory olfactory neurons, continually undergoes neurogenesis and axonal outgrowth throughout life. We describe here several new olfactory system-specific glycoforms of the neural cell adhesion molecule N-CAM in the frog, R. catesbeiana. Using immunochemical methods for in situ localization, we show that the lectin dolichos biflorus agglutinin (DBA) and two monoclonal antibodies, 9OE and 3A6, detect three unique N-CAM forms present on primary sensory olfactory axons. In addition, DBA and monoclonal antibody 9OE recognize glycoconjugates and/or N-CAM glycoforms expressed specifically in discrete central olfactory pathways and regions in frog brain. This is a novel example of unique adhesion molecule forms present in a chain of two neurons within a vertebrate neural pathway. Together these glycoconjugates and N-CAM glycoforms may participate in cellular interactions associated with olfactory system pathway formation and renewal.

Immunochemical markers for the frog olfactory neuroepithelium.

Monoclonal antibodies (mAbs) were generated that react with the major cell types in the olfactory neuroepithelium of the frog, Rana catesbeiana. This pseudostratified epithelium consists of apical supporting cells, a middle layer of olfactory receptor neurons and a heterogeneous population of basal cells consisting of basal cells proper and globose basal cells. Both olfactory receptor neurons and globose basal cells were labelled by mAb 13-OE, which recognized the neural cell adhesion molecule NCAM. The identity of these NCAM positive cells was established by analysing regenerating olfactory epithelium and by a double-antibody labelling immunofluorescence technique. The olfactory nerve was lesioned, which induced the death of olfactory receptor neurons and the subsequent proliferation of basal cells. When the regenerating olfactory epithelium was analysed prior to the reconstitution of mature olfactory neurons, mAb 13-OE reacted specifically with globose basal cells and not the basal cells proper. Simultaneous labelling of normal olfactory epithelium with mAb 13-OE and polyclonal anti-keratin antibodies, the latter of which labels supporting cells and basal cells proper, revealed no double-labelled cells. These results further confirmed that NCAM was expressed by both globose basal cells and receptor neurons but not by other cell types within the epithelium. Additional cell types in the olfactory epithelium reacted with other new mAbs: 4-OE, 5-OE, 7-OE and 9-OE. Supporting cells were stained by mAb 4-OE. Olfactory receptor neurons and the entire population of basal cells were immunoreactive with mAb 7-OE. The cilia and knobs of receptor neurons were strongly immunoreactive with mAb 5-OE whereas mAb 9-OE selectively stained olfactory knobs and not the cilia on these chemosensory cells. These studies are a first step towards experimental approaches designed to elucidate the mechanisms underlying the unique proliferative properties of the olfactory neuroepithelium in frog.

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM).

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.

Rat olfactory cells and a central nervous system neuronal subpopulation share a cell surface antigen.

Using the monoclonal antibody (Mab) 6B7, a cell surface component found in adult rat central nervous system membrane preparations and on the surfaces of a subpopulation of neurons in cultures of embryonic rat forebrain has been identified. This Mab was derived from mice immunized with a rat forebrain synaptic plasma membrane preparation. High levels of Mab 6B7 binding are observed with membrane preparations from rat forebrain and olfactory bulb but no detectable binding is observed with membranes from the non-neural adult rat tissues heart, kidney, liver, lung and testes. Binding to dorsal root ganglia preparations was 5-fold lower than to forebrain. In immunofluorescence analyses, Mab 6B7 binds to the surface of a significant proportion of neurons in cultures of embryonic day 14 rat forebrain. However, it is absent from GFAP-positive astrocytes, oligodendrocytes, Schwann cells and fibroblastic cells in rat neural cultures. Due to the high levels of binding in olfactory tissue, the distribution of the 6B7 antigen in the olfactory epithelium was characterized in greater detail. In cryostat sections, 6B7 appears to react with a cell population of the basal layer of the adult rat epithelium, but is absent from the cell bodies of the more mature neuronal population which lies higher in the epithelium. This result suggests that within the olfactory epithelium Mab 6B7 may be useful as a marker for the proliferative basal cells which are the neuronal precursors in the epithelium. In summary, the 6B7 antigen may be useful in identifying and analyzing cell subpopulations in both the central nervous system and olfactory epithelium.

Polypeptide variation in an N-CAM extracellular immunoglobulin-like fold is developmentally regulated through alternative splicing.

The alternative splicing of a previously undiscovered 30 base exon confers a new level of polypeptide diversity on the N-CAM family of cell-surface glycoproteins. It results in the insertion of 10 amino acids into the fourth of five extracellular immunoglobulin-like folds. Each major size class of rat brain N-CAM mRNAs consists of members that contain or lack the exon. Furthermore, this splicing event is developmentally controlled: RNAs containing the inserted exon are expressed at extremely low levels (less than 3%) in embryonic brain but increase postnatally to 40%-45% of all N-CAM mRNAs in adult brain. Antibodies that recognize the alternative 10 amino acid segment react with a subset of N-CAM-expressing neurons in cultures of embryonic rat cells.

Smooth muscle cells transiently express NCAM.

NCAM (neural cell adhesion molecule) polypeptides were first detected on neuronal cells and were subsequently found to be expressed at least transiently by a number of other cell types including skeletal and cardiac but not smooth muscle. We report here that rat smooth muscle expresses NCAM in vitro and transiently in vivo. Using a monoclonal antibody 3F4 which reacts with most rat NCAM polypeptides, NCAM was found on the surface of cultured rat aortic smooth muscle lines A10 and A7r5 and mouse smooth muscle like line BC3H1 in abundances equal to or greater than those of cardiac muscle, skeletal muscle, and neuronal cell lines. The major NCAM polypeptide of muscle cells was Mr = 140 kDa with lesser amounts of the 120 kDa form. Consistent with these results, a major NCAM mRNA of 6.7 kb was detected in Northern analyses with lesser amounts of the 4.3 and 2.9 kb mRNA size classes. The relative abundance of NCAM mRNA was similar in RNA prepared from smooth muscle A7r5 cells, L6 skeletal muscle cells, and 9-day-old rat brain. NCAM was distributed across the entire surface of cultured smooth muscle cells in a highly punctate manner. Cryostat sections of rat aorta, intestine and bladder were examined by immunofluorescence to determine if NCAM is also expressed on smooth muscle in vivo. In each case NCAM was found to be transiently expressed by the smooth muscle cells of these tissues. Highest NCAM levels were observed at embryonic day 17 which then declined to undetectable levels in tissues from adults. These results extend previous observations to indicate all muscle types transiently express NCAM in development.

Extracellular matrix derived from astrocytes stimulates neuritic outgrowth from PC12 cells in vitro.

The ability of astrocyte extracellular matrix to stimulate axonal elongation was examined using an in vitro model system. Extracellular matrix (ECM) was derived from primary cultures of rat astrocytes or meningeal cells, or from a cell line of bovine aortic endothelial cells. The cells were grown in 35-mm tissue culture dishes for 24 h and then removed non-enzymatically, leaving ECM attached to the surface of the culture dishes. Subsequently, PC12 pheochromocytoma cells were seeded onto the ECM and de novo neurite outgrowth was measured. Within 24 h, the PC12 cells exhibited profuse neuritic outgrowth on ECM derived from astrocytes and endothelial cells, without addition of exogenous nerve growth factor. Over a period of 4 days, the neurites continued to elongate and branched extensively. Little or no neuritic outgrowth was observed from PC12 cells grown on uncoated culture dishes or on culture dishes treated with astrocyte-conditioned medium. Only a slight stimulation of neurite outgrowth was observed on meningeal cell-derived ECM. These results indicate that astrocyte ECM, as well as endothelial cell ECM, possesses one or more molecular factors that can stimulate and maintain de novo axonal elongation from PC12 cells. It is suggested that immature astrocytes secrete neurite-promoting factors as a component of the ECM which act to stimulate and possibly guide the growth of axons during in vivo development.

Differential redistribution of lectin receptor classes on clonal rat myotubes and myoblasts.

To evaluate the relative mobilities of cell surface glycoconjugates during myogenesis we have studied the redistribution of fluorescein-conjugated plant lectins on L6 rat myogenic cells. Previous experiments had demonstrated that the receptors for the lectins soybean agglutinin (SBA), wheat germ agglutinin, concanavalin A and Lens culinaris agglutinin all were relatively uniformly distributed on both myoblasts and myotubes, and that SBA receptors were capable of rapid redistribution on myotubes but not myoblasts at 4 degrees C (Sawyer & Akeson, 1983). Here we show that when SBA-labelled myoblasts are incubated at 37 degrees C, or for extended times at 4 degrees C, the lectin aggregates as on myotubes. So it appears that SBA-binding components show a quantitative rather than qualitative change in their mobility during L6 differentiation. In addition, the redistribution of the three other lectins on myoblasts and myotubes was either less prominent (i.e. showing fewer apparent surface clusters) or occurred less rapidly than with SBA. None of these three lectins showed striking differences in mobility between myoblasts and myotubes. Thus, it appears that SBA binds to a subset of surface glycoconjugates that is relatively highly mobile, and that this mobility is specifically enhanced with differentiation.

Cell-surface antigens of developing rat cerebellar neurons: identification with monoclonal antibodies.

Three monoclonal antibodies (Mab's), named 3C5.59, 3G5.34, and 3G6.41, that recognize cell-surface antigens found on embryonic and postnatal neurons were selected for study from among a group generated against adult rat synaptic plasma membranes (SPM's). Immunofluorescence staining with these 3 antibodies showed strong reactivity of the processes and much weaker staining of the cell bodies of the small neurons cultured from early postnatal rat cerebella. Mab 3G6.41 also reacted with flat astrocyte-like cells cultured from cerebellum. In contrast Mab's 3C5.59 and 3G5.34 appeared to specifically recognize only the neurons in these cultures. In situ staining of cryostat sections with 3G5.34 and 3G6.41 demonstrated immunoreactivity that was predominantly localized to the molecular layer of the cerebellum in the early postnatal through adult stages, in agreement with the strong staining of neurites seen in primary neuronal cultures. Quantitative analysis of Mab binding to particulate protein preparations from various tissues of the adult rat indicated that each of the antigens is restricted to the nervous system in the adult. Binding studies also indicated that each antigen was enriched approximately 1.5-fold in the SPM fractions compared to total particulate fractions from cerebellum. All 3 Mab's recognize membrane-bound molecules that can be solubilized by non-ionic detergent. Mab 3G6.41 immunoprecipitated two polypeptides of 140,000 and 185,000 apparent molecular weight from detergent-solubilized cerebellar cells that were surface-iodinated in culture. These antibodies should prove useful in the further analysis of the expression and function of individual cell surface antigens during the differentiation of cerebellar granule cells.

Clonal myoblasts and myotubes show differences in lectin-binding patterns.

To determine changes in distribution or mobility of cell-surface glycoconjugates during myogenesis the binding of fluorescein-conjugated plant lectins to myoblasts and myotubes of the L6 rat skeletal muscle cell line has been studied. Binding has been carried out at 4 degrees C on either live or glutaraldehyde-fixed cells. Fluorescein conjugates of soybean agglutinin (Fl-SBA), wheat germ agglutinin (Fl-WGA), concanavalin A (Fl-conA) and Lens culinaris agglutinin (Fl-LCA) produced predominantly uniform fluorescence on both live and fixed myoblasts. On fixed myotubes, Fl-LCA, Fl-conA and Fl-SBA again produced predominantly uniform fluorescence, whereas Fl-WGA showed a pattern of diffuse, irregular spots in addition to uniform fluorescence. Fl-conA, Fl-LCA and Fl-WGA binding to live myotubes resulted in patterns quite similar to those on fixed myotubes; the only differences being the presence of weak patterns of diffuse spots with Fl-LCA and Fl-conA and an enhanced pattern of diffuse spots with Fl-WGA. Fl-SBA, however, showed a unique pattern on live myotubes which consisted of discrete, round spots and minimal uniform fluorescence. With shorter labeling times, Fl-SBA produced relatively more prominent uniform fluorescence on live myotubes. It appears, therefore, that the native distribution of SBA, conA and LCA-binding sites is similar and predominantly random on L6 myoblasts and myotubes, whereas some WGA-binding sites may be aggregated on myotubes. The results also suggest that SBA-binding sites readily cluster at 4 degrees C on myotubes but not myoblasts, whereas the other lectin sites undergo little or no redistribution on either cell type. Thus the mobility of SBA-binding sites may increase with differentiation.

Induction of expression of the rat G5 nervous system antigen occurs postnatally.

G5 is a cell membrane component found in high levels in the adult rat central nervous system and in low levels on some cells of the rat immune system. Binding assays with adult brain particulate protein preparations and monoclonal antibody to G5 (G5-IgG) gave highest activity in cerebral cortex and lowest in the white matter rich regions pons and spinal cord. Three peripheral nervous system components had no G5 activity. Analysis of G5 content in particulate protein preparations from whole rat brain of ages embryonic day 15 to adult indicates that G5 is present in negligible amounts in the newborn rat. It increases in both specific and total activity to reach adult levels by postnatal day 30. A similar induction curve was observed with cerebellum samples. In adult cerebellum, high levels of G5 were found in the molecular layer using both autoradiographic and immunofluorescence techniques. White matter had essentially no activity. The density and uniformity of reaction product in these techniques suggest that G5 is present on a major cellular constituent of the molecular layer. Pharmacologic experiments indicate G5 is not detectable on climbing fibers or adrenergic fibers. Cerebellar samples from juvenile rats also had predominant G5 activity in the forming molecular layer.