Maintenance of rat taste buds in primary culture.

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.

Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses.

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Na(v)1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Na(v)1.6-deficient mouse mutant strain med, whose nodes were negative for Na(v)1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Na(v)1.6 is the predominant channel type in this structure. In the central nervous system, Na(v)1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Na(v)1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

ETO and AML1 phosphoproteins are expressed in CD34+ hematopoietic progenitors: implications for t(8;21) leukemogenesis and monitoring residual disease.

To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ ETO protein in normal hematopoiesis and in leukemia, we raised rabbit antisera to a bacterially expressed polypeptide containing amino acid residues 1 to 220 of ETO and to synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross-reactivity with other members of the PEBP2 alpha family. With affinity-purified reagents, we observed immunofluorescent staining for both AML1 and ETO in the nucleus of HEL, K562, and Kasumi-1 leukemic cell lines, the last from a t(8;21) AML. Biochemical analysis confirmed specificity of the antibodies and the nuclear localization of the antigens, the latter being exclusive for AML1 and primary for ETO. Immunoprecipitations of metabolically labeled 32P-proteins from Kasumi-1 cells show that AML1 and ETO are phosphorylated on serine and threonine. Investigations with normal bone marrow reveal AML1 and ETO are coexpressed in megakaryocytes and that each is expressed in a portion of the approximately 10-microns-diameter cells residing there. Using a CD34+ enriched population mobilized to peripheral blood, we found AML1 and, unexpectedly, ETO present in these cells. Because of this, we conclude that the expression of ETO in hematopoietic cells is not by itself leukemogenic. Also, because ETO would not be exclusively expressed as part of chimeric AML1/ETO in leukemic patients, its presence cannot be used to monitor t(8;21) AML residual disease.

The putative membrane anchor protein for yeast Sec7p recruitment.

Proteins required for yeast secretory pathway function have been identified by genetic selection and characterization of the temperature-sensitive secretory (sec) mutants. The use of genetic and biochemical approaches has expanded the catalog of components of the secretory pathway, yet many proteins, especially membrane and lumenal proteins, remain to be identified. Sec7p, one of the original SEC gene products to be described, is required at multiple stages of the yeast secretory pathway in the coating of transport vesicles. A chemical cross-linking approach was used to identify proteins associated with Sec7p protein complexes from yeast cell lysates. A 90 kDa integral membrane protein (p90) was isolated whose interactions with Sec7p were reproduced in the absence of chemical cross-linking. Further biochemical analysis indicated that p90 may act as the anchor protein for Sec7p membrane recruitment in transport vesicle assembly.

A developmentally regulated plasmalemmal antigen present in synaptosomes but not in growth cones.

Monoclonal antibody 2L4 was generated against rat synaptosomes but does not cross-react with nerve growth cones. Expression of the 2L4 antigen is developmentally regulated in a manner that is, in part, the opposite of the expression of the 5B4-CAM antigen, a marker of neuronal outgrowth belonging to the N-CAM family. While 5B4-CAM appears and increases during sprouting and then decreases to reach low levels in the adult, the 2L4 antigen appears only late in development, when neuronal outgrowth ceases, at or around the time of synaptogenesis. Once expressed, the antigen is found on the entire plasmalemmal surface of the neuron, but seems to be enriched at synaptic endings, at least of some neuron types. Biochemical analyses involving blotting of non-denaturing gels and immunoaffinity chromatography identify the antigen as a pair of polypeptides with similar, basic isoelectric points. These polypeptides form a somewhat diffuse, probably glycosylated band at 67 kDa and may be part of a hetero-oligomeric complex. The localization, biochemical, and developmental results suggest that the 2L4 antigen is a plasmalemmal marker of maturing and/or mature neurons whose expression may be triggered by synaptogenesis.

Immunocytochemical localization of endopeptidase-24.11 in the nucleus tractus solitarius of the rat brain.

Recently, it has been hypothesized that the N-terminal portion of substance P (SP), SP(1-7), which results from the action of endopeptidase 24.11 (EC3.4.24.11), could be involved in mediating the depressor effects of baroreceptor afferent activation via its action on cells in the nucleus tractus solitarius (NTS). In this study, the binding of a monoclonal antibody to endopeptidase 24.11 was examined immunohistochemically at the level of the caudal medulla of the rat brain. By light microscopy, intense immunoreactivity was seen in the NTS, in fibers bordering the area postrema, and in the area postrema itself. After electron microscopy, endopeptidase 24.11-like immunoreactivity was seen to be associated with the cytoskeleton and plasma membrane in axons, dendrites and glial processes. Antigen was also associated with synaptic vesicles and plasma membranes in presynaptic terminals forming mainly axo-dendritic synapses typical of vagal afferent terminals involved in the baroreceptor reflex. Thus, endopeptidase 24.11 appears to be localized at sites where it could effectively process SP prior to its binding to postsynaptic receptors.

A monoclonal antibody against the slow isoform of skeletal muscle Ca2+-ATPase selectively stains a subpopulation of neurons in the central nervous system.

A monoclonal antibody against the slow isoform of chicken skeletal muscle Ca2+-ATPase recognises, in nervous tissue, analogous membrane proteins which are most concentrated in the microsomal fraction. Histochemically, the immunoreactivity of the antibody is restricted to neurones where the staining is most intense in cell bodies and dendrites, weak in axons and absent from cell nuclei. The expression of the antigen varies greatly between different neuronal populations and is developmentally regulated. The antigen is also axonally transported.

The binding of a monoclonal antibody reactive with pp60v-src to the rat CNS both in vitro and in vivo: evidence that the epitope is present intracellularly as well as being associated with a number of antigenically related polypeptides located externally in the plasma membrane only in the synaptic region.

A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects.

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.

Association of synapsin I with neuronal cytoskeleton. Identification in cytoskeletal preparations in vitro and immunocytochemical localization in brain of synapsin I.

Calmodulin-dependent protein kinase II (CaM kinase II) is associated with microtubule preparations and phosphorylates several endogenous proteins including microtubule-associated protein 2, tubulin, and an 80,000-dalton protein doublet (pp80). We now report that pp80 is identical to synapsin I by all criteria studied including molecular weight, isoelectric point, phosphopeptide mapping of cAMP- and calmodulin-dependent phosphorylated protein, comigration with authentic synapsin I, and sensitivity to digestion with collagenase. Synapsin I and CaM kinase II were found in association with both microtubule preparations and preparations enriched in neurofilaments. Antibodies to synapsin I specifically labeled neurofilaments prepared in vitro. Immunocytochemical studies on rat brain tissue demonstrated synapsin I immunoreactivity specifically associated with the neuronal cytoskeleton as well as synaptic vesicles. The observed synapsin I staining on cytoskeletal elements was considerably diminished or abolished by the inclusion of Triton X-100 in the staining solutions. These results indicate that synapsin I is associated with the cytoskeleton and may be an important link between cytoskeletal elements as well as between the cytoskeleton and membrane.

Immunocytochemical localization of serotonin in the rat dentate gyrus following raphe transplants.

The ultrastructural features of the serotoninergic innervation of the rat dentate gyrus in normal adults and in animals receiving raphe nuclear area transplants was investigated using an antibody to serotonin (5-HT). Neonatal rats received a lesion of the fimbria-fornix and entorhinal cortex. Three days later, a portion of embryonic (E-16-18) raphe nuclear area was transplanted to the entorhinal cavity and the animals were allowed to survive for 60 days. Animals were processed for the immunocytochemical localization of 5-HT using the peroxidase-antiperoxidase method. Light microscopic observation showed that 5-HT-containing fibers from transplanted raphe neurons densely innervated the hilar and molecular zones of the dentate gyrus. Electron microscopic analysis showed that 5-HT immunoreactivity was contained only in axons and axon varicosities. There were no differences in the ultrastructural characteristics of axons and axon terminals between normal animals and those which had received raphe transplants. A mixture of both conventional synaptic junctions and non-synaptic axonal swellings were found in both groups.

Association of calcium/calmodulin-dependent kinase with cytoskeletal preparations: phosphorylation of tubulin, neurofilament, and microtubule-associated proteins.

Calcium and calmodulin have been implicated in the regulation of cytoskeletal function. In this report, we demonstrate that microtubule preparations from rat brain contain a calcium/calmodulin-dependent protein kinase that phosphorylates endogenous MAP-2, tubulin, synapsin I, and neurofilament proteins. This cytoskeletal-associated kinase has been biochemically characterized and shown to be identical to Type II calcium/calmodulin-dependent protein kinase (CaM kinase II). The subunits of CaM kinase II represented major calmodulin-binding proteins in cytoskeletal preparations. A monoclonal antibody against the 52000 Da subunit of CaM kinase II specifically labeled cytoskeletal elements in cortical neurons. These results indicate that CaM kinase II is associated with the neuronal cytoskeleton and may play a role in mediating some of the effects of calcium on cytoskeletal function.

Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens.

Polypeptides (233 micrograms) from brain synaptosomes resolved in SDS 7.5%--15% polyacrylamide gradient gels were electrophoretically transferred quantitatively to nitrocellulose sheets at 3 V/cm for 21 h in the following buffer: 25 mM Tris-192 mM glycine, pH 8.3/20% methanol/0.1% SDS. After immunoautoradiographic detection of antigens on this nitrocellulose replica with either rabbit anti-rat cerebrum immunoglobulins or a mouse monoclonal antibody to a rat synaptosomal protein, the antibody and [125I]protein A were removed from the replica by treatment with 8 M urea, 0.1 M 2-mercaptoethanol, and 5 mg/ml BSA at 60 degrees C for 1 h. The replicas were successfully re-used by re-exposing them to the antibody and [125I]protein A.

Distribution of fucosyl-conjugates in rat cerebellar cells in vitro: binding patterns of Lotus tetragonolobus and Ulex europeus lectins.

Light microscope radioautographic procedures were employed to investigate the binding patterns of lectins derived from Lotus tetragonolobus (Lotus A) and Ulex europeus (UEA I) to the surfaces of rat cerebellar cells maintained in dispersed cell culture. Lotus A bound extensively to the surfaces of neuronal cell somata and neuronal processes at all time points investigated; the lectin also bound to nonneuronal cell surfaces, but to an extent much less than that for neurons. Lectin binding was completely inhibited by the presence of 0.1 M alpha-L-fucose in the reaction medium. Conversely, UEA I did not appear to bind to any significant extent to the surfaces of cerebellar cells in vitro at any stage of maintenance. The incorporation of [3H]fucose into fucosyl-conjugates by cerebellar neurons in culture was investigated using light microscope radioautography and polyacrylamide gel electrophoretic methods. Radioautographs indicated that the fucose came to be distributed throughout neuronal cell somata and processes and that, with increasing time, the grain density appeared to be highest over areas of neuropil and fascicles of cellular processes. The electrophoresis data demonstrated that, among others, the label was incorporated into prominent classes of polypeptides having nominal molecular weights of 400,000, 125,000, 91,000 and 46,000 daltons.

Distribution of the LETS protein (fibronectin) in rat cerebellum. An in vitro and in vivo developmental study.

The distribution of the large, external, transformation-sensitive (LETS; fibronectin) protein was investigated in rat cerebellum, both in vitro and in vivo, by biochemical and immunocytochemical methods. Biochemical analyses indicated that LETS protein is not demonstrable on the surfaces of cerebellar neurons from postnatal rats maintained in cell culture for varying periods of time, but is present on the surfaces of at least some fraction of the total nonneuronal cell population in vitro. Indirect immunofluorescence microscopy with an anti-LETS antiserum substantiated these observations and further indicated that LETS-bearing cells of cerebellum maintained in vitro are probably of endothelial and fibroblastic origin. The LETS protein is arranged in a reticular network of filaments spanning the surfaces of the cells, and the filaments are often extensively interdigitated with each other. At all stages of development investigated (two days postnatal to adult) LETS antigen was observed in vivo to be primarily localized in the meninges covering the surface of the cerebellum and between folia, and in the walls of blood vessels within the tissue. Neuroblasts and neurons of the external and internal granule layers of the cerebellum, respectively, were negative for the presence of LETS antigen.

Cell surface proteins of differentiating rat cerebellar cells maintained in dispersed cell culture.

The complement of cell surface proteins of differentiating rat cerebellar neurons maintained in dispersed cell cultures was evaluated by the combined methods of lactoperoxidase-catalyzed radioiodination and miniature sodium dodecyl sulfate polyacrylamide gel electrophoresis. Pure neuronal cell samples were obtained by direct dissection of such cells from the surfaces of the culture vessels. The electrophoretic profiles of cell surface proteins (CSP) from neurons maintained for 1, 7, 14, 21 and 28 days in vitro (DIV) were compared. It could be seen that while the pattern was relatively constant between 7 and 28 DIV, several differences existed between profiles from 1 and 7 DIV. The earliest stage was characterized by a prominent polypeptide triplet having molecular weight of approximately 48,000, 56,000 and 71,000 daltons. Later stages of development (7-28 DIV) were characterized by a greater number of high molecular weight surface polypeptides relative to the earliest stage. Comparison of electrophoretic profiles of CSPs of neurons and nonneuronal cells indicated that a number of polypeptides were not shared by these classes of cells.

Axonal transport of the Ca2+-dependent protein modulator of 3':5'-cyclic-AMP phosphodiesterase in the rabbit visual system.

Water-soluble proteins were extracted from individual retinas, optic nerves, combined optic tracts and lateral geniculate bodies, and superior colliculi of rabbits at 1, 3, and 18 days after injection of [3H]leucine into the right eye. The Ca2+-dependent protein modulator of 3':5'-cyclic-AMP phosphodiesterase (calmodulin) was isolated from these samples by a two-step polyacrylamide gel electrophoresis procedure. An analysis of the radioactivity incorporated into the total soluble proteins and the calmodulin revealed that most of the calmodulin was axonally transported at a slow rate (2--4 mm/day) and represented about 0.45% of the total transported soluble protein.

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography.

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.