Biomedical subjects
J I Morgan
Publications and source records attributed to J I Morgan.
The neuropeptide cerebellin is a marker for two similar neuronal circuits in rat brain.
We report here that the neuropeptide cerebellin, a known marker of cerebellar Purkinje cells, has only one substantial extracerebellar location, the dorsal cochlear nucleus (DCoN). By reverse-phase high-performance liquid chromatography and radioimmunoassay, cerebellum and DCoN in rat were found to contain similar concentrations of this hexadecapeptide. Immunocytochemistry with our rabbit antiserum C1, raised against synthetic cerebellin, revealed that cerebellin-like immunoreactivity in the cerebellum is localized exclusively to Purkinje cells, while in the DCoN, it is found primarily in cartwheel cells and in the basal dendrites of pyramidal neurons. Some displaced Purkinje cells were also stained. Although cerebellum and DCoN receive their inputs from different brain regions, their organizations show several similarities, with many neuronal cell types present in one having their presumed homologue in the other. Cerebellin is present in Purkinje and cartwheel cells throughout the cerebellum and the DCoN without site predilection. This should be viewed together with recent data indicating that these two classes of neurons share a similar fate in several murine mutations ("nervous," "lurcher," "Purkinje cell degeneration," and "staggerer"). This situation leads us to conclude that cerebellin represents a marker for a small number of spatially related neuronal cell classes, and that it may play a role in an as yet unknown aspect of a circuit function worthy of further exploration.
Monoclonal antibodies to mammalian carnosine synthetase.
A set of mouse monoclonal antibodies has been generated against rabbit muscle carnosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross-reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low-abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.
The polypeptide PEP-19 is a marker for Purkinje neurons in cerebellar cortex and cartwheel neurons in the dorsal cochlear nucleus.
This light and electron microscopic immunocytochemical study shows that the polypeptide PEP-19, a presumptive calcium binding protein specific to the nervous system, represents an excellent marker for cerebellar Purkinje cells and dorsal cochlear nucleus (DCoN) cartwheel cells. The polypeptide clearly reveals the entire populations of both types of neurons, including their complete dendritic and axonal arborizations. Other PEP-19 containing neurons in the two regions display weak immunoreactivity restricted to the cell body or to cell body and principal dendrites. Electron microscopic localization of PEP-19-like immunoreactivity reveals similarities between this polypeptide, parvalbumin, and a 28K vitamin D-dependent calcium binding protein. However, calmodulin, which is expressed in both Purkinje and granule cells, may differ from PEP-19. Similarities between the organization of the cerebellar cortex and the DCoN superficial layers have been known for some time, with several types of neurons in one system having their presumed homologue in the other. These data provide further support for the proposed structural and functional homology between Purkinje and cartwheel neurons, and establishes PEP-19 as a useful marker for examining degeneration of these two neuronal populations in murine cerebellar mutants.
Isolation, sequence, and developmental profile of a brain-specific polypeptide, PEP-19.
By comparing the HPLC profiles of cerebellar extracts from adult and neonatal rats, a developmentally regulated polypeptide, termed PEP-19, was identified. The concentration of PEP-19 rose from 0.1 nmol/g of cerebellum at birth to 2 nmol/g at 20 days postpartum. The polypeptide could also be detected at lower levels in olfactory bulbs of adult rats but was absent in cerebral cortex, brain stem, and all non-neural tissues examined. HPLC-purified PEP-19 contained 61 amino acids and had a molecular size of 7.6 kDa. The native polypeptide is blocked at its amino terminus but was sequenced following proteolytic and chemical fragmentation. The primary amino acid sequence was determined to be: X (S-E) R Q S A G A T N G K D K T S G D N D G Q K K V Q E E F D I D M D A P E T E R A A V A I Q S Q F R K F Q K K K A G S Q S. PEP-19 has a unique sequence, but shares some homology with several calcium binding proteins including the beta chain of S100 and intestinal calcium binding protein. This polypeptide is the primary translation product of cerebellar poly(A)+ mRNA.
Barium modulates c-fos expression and post-translational modification.
The addition of exogenous barium ions to PC12 rat pheochromocytoma cells elicits a transient induction of the c-fos gene. Induction is antagonized by extracellular calcium and the dihydropyridine calcium channel blockers, and it is attenuated in the presence of calmodulin inhibitors. Thus, barium appears to enter the cell through a voltage-dependent calcium channel and, either directly or indirectly, interacts with calmodulin to stimulate c-fos expression. This property of barium is not shared by a range of di- and trivalent cations examined. Agents that induce the c-fos gene in PC12 cells may be divided into two broad categories. The first comprises polypeptide growth factors and phorbol esters, which give rise to a c-fos protein that undergoes extensive post-translational modification. The second, which comprises depolarizing agents and barium ions, acts via calcium channels and yields a c-fos protein that undergoes less extensive post-translational modification. These different forms of c-fos protein can be distinguished on the basis of their apparent molecular weights on sodium dodecyl sulfate/polyacrylamide gels.
Downregulation of [3H]Ro5-4864 binding sites after exposure to peripheral-type benzodiazepines in vitro.
Peripheral-type benzodiazepine (BZD) binding sites undergo a rapid and pronounced downregulation after exposure to these compounds in vitro. Friend erythroleukemia cells were incubated with micromolar concentrations of BZD after which they were washed thoroughly and the binding of the specific peripheral-type BZD radioligand [3H]Ro5-4864 was determined. Exposure to the peripheral-type BZD Ro7-3351 decreased the number of [3H]Ro5-4864 binding sites from 324 to 41 fmol/10(6) cells with no change in affinity. Downregulation appears to require active cellular processes because it is blocked when exposure to BZD is at 4 degrees C rather than at 37 degrees C. Furthermore, whereas [3H]Ro5-4864 binding is decreased substantially in membrane preparations made from downregulated cells, it is not altered when membrane preparations from control cells are exposed to BZD. The time course of downregulation is quite rapid, as it occurs within minutes. In contrast, the return of sites requires days and there is a close relationship between return of sites and growth of new cells. The ability of BZDs to downregulate correlates more closely with affinity for the peripheral-type site than with biological activity. The ability to undergo downregulation is characteristic of receptors and its occurrence suggests that peripheral-type BZD binding sites are functional receptors.
Vitamin D3 derivatives inhibit the differentiation of Friend erythroleukemia cells.
A number of vitamin D3 metabolites inhibit benzodiazepine- and dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. The inhibition is dose dependent and occurs at nM concentrations. The order of potency of these compounds is 1,25-dihydroxycholecalciferol greater than 1,25,26-trihydroxycholecalciferol greater than 1,24R,25-trihydroxycholecalciferol greater than 1 alpha-hydroxycholecalciferol greater than 24R,25-dihydroxycholecalciferol greater than 25S,26-dihydroxycholecalciferol. The inhibition is maximal when the vitamin D3 analogs are added together with the inducer, and becomes progressively decreased with delayed addition. These results suggest that the vitamin D3 metabolites may play a regulatory role in erythropoiesis.
Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines.
Alterations in proto-oncogene expression after stimulation of rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) have been investigated. A specific stimulation of c-fos messenger RNA and protein was detected 30 minutes after treatment. This induction was enhanced more than 100-fold in the presence of peripherally active benzodiazepines. The effect was specific as very little change was observed in the levels of c-rasHa, c-rasKi, c-myc, and N-myc messenger RNA's. Under the conditions used here, NGF treatment ultimately results in neurite outgrowth, with a reduction or cessation of cell division. Thus, stimulation of the c-fos gene in this system appeared to be associated with differentiation and not with cellular proliferation. The effect of benzodiazepines was stereospecific and represents a novel action of these compounds at the level of gene expression.
Peripheral-type benzodiazepines influence ornithine decarboxylase levels and neurite outgrowth in PC12 cells.
A number of the benzodiazepines (BZDs) inhibit nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner in PC12 cell cultures. The rank order of potency of a series of BZDs for inhibition of neurite outgrowth does not correlate with the order of their affinity constants for the so-called peripheral BZD sites present on PC12 cells. Whereas the inhibition of neurite extension is stereospecific, the binding to the peripheral site is not. The inhibition of neurite outgrowth is not attributable to a blockade of NGF binding to either its fast or slow receptors. Additionally, several other characteristics of NGF stimulation of PC12 cells, such as autoadhesion and the induction of ornithine decarboxylase (OrnDCase), remain unaltered in the presence of BZDs. Many BZDs increase OrnDCase levels in PC12 cells in the absence of NGF. The OrnDCase response to BZDs is blocked by actinomycin D. Furthermore, the structural requirements of BZDs for induction of OrnDCase activity is not identical to that for the inhibition of NGF-induced neurite extension. Thus, unlike attenuation of neurite extension, BZD induction of OrnDCase is not stereospecific. Also, some BZDs block neurite growth but do not induce OrnDCase. We propose that there are at least three sites of action of BZDs on PC12 cells. The first is the well-characterized high-affinity peripheral site. The second distinct locus of action results in a blockade of NGF-induced neurite outgrowth. The structural requirements for the latter effect are essentially indistinguishable from those for BZD induction of hemoglobin synthesis in Friend erythroleukemia cells. The third site of action results in an induction of OrnDCase. Structural requirements for this activity are not identical to those for either the differentiation of Friend cells or the inhibition of neurite outgrowth.
Cerebellin: a quantifiable marker for Purkinje cell maturation.
The cerebellum-specific hexadecapeptide cerebellin has been localized by immunocytochemical means to the perikarya and dendrites of cerebellar Purkinje cells. Biochemical analysis using ion-pairing HPLC shows cerebellin to first appear 5 days after birth, whereafter levels rise to a maximum at 25 days postpartum, and then decline to stable adult values. This same pattern of development occurs with a lag of approximately 5 days for the major metabolite of cerebellin, des-Ser1-cerebellin. The immunocytochemical picture of cerebellin in developing Purkinje cells mirrors the biochemical data. These results show that cerebellins represent unique quantifiable markers for the investigation of Purkinje cell maturation and lend support to the feasibility of using unique endogenous peptides to chart neurodevelopment.
A panel of monoclonal antibodies to the rat olfactory epithelium.
BALB/c mice were immunized with dissected olfactory mucosa from young adult Sprague-Dawley-derived, CD, rats and antibody-secreting hybridomas were produced. Supernatants from hybridoma cultures were screened by immunocytochemical methods for their ability to react with specific cell populations in frozen sections of rat olfactory epithelium. Approximately 60 clones were identified that showed various degrees of specific staining. These have been classified on the basis of their particular staining specificities. One group of monoclonal antibodies, designated LUM, reacts with the luminal surface of the epithelium. Closer examination reveals these antibodies to react variously with the apical brush border of sustentacular cells, respiratory cilia, and the luminal membrane of respiratory cells. Another group of monoclonal antibodies reacts primarily with sustentacular cells, as is indicated by the SUS prefix. Some antibodies in this group also react with the membranes of respiratory cells and the cells comprising the acini of Bowman's glands. A larger group of antibodies reacts with olfactory neurons and/or their axons, as denoted by the NEU prefix. This group can be further subdivided by the criteria of whether both the olfactory nerve and vomeronasal nerve react with the same monoclonal antibody. A fourth group of monoclonal antibodies, designated GLA, reacts with Bowman's glands and in some instances with secretory cells present in the respiratory mucosa. Two clones, BCL, stain at the level of the basal cell layer just above the lamina propria. A number of other antibodies react with cells and structures of the epithelium that have not been previously described. One of this group, NIS-1, stains globular structures present in the mucosa of the neuroepithelium.
Monoclonal antibodies reveal novel aspects of the biochemistry and organization of olfactory neurons following unilateral olfactory bulbectomy.
Following unilateral olfactory bulbectomy in rats the ipsilateral olfactory neuroepithelium undergoes degeneration. Subsequently, the receptor neuron complement of the tissue is restored by the proliferation and differentiation of immature neuroblasts. However, as noted by other workers, in the absence of a target organ the dynamics of neuron regeneration is altered such that there is an overall reduction in the number of cells positive for the olfactory marker protein when cellular equilibrium is re-established. Immunocytochemical staining of the olfactory epithelium of unilaterally bulbectomized rats with a series of anti-neuronal monoclonal antibodies reveals an attenuation of binding of some antibodies to the neurons of the ipsilateral epithelium. In contrast, other anti-neuronal monoclonal antibodies show no difference in staining intensity when ipsilateral and control contralateral epithelia are compared. These data suggest that the expression of some neuronal antigens is subject to control by the target olfactory bulb, whereas others are independent of such putative regulation. Besides altering the expression of some antigenic determinants, olfactory bulbectomy also results in certain organizational changes in epithelium. First, bulbectomy produces an increase in the incidence of a cell type that appears to span the neuroepithelium. Although the morphology of these cells is more akin to a sustentacular cell than to a receptor neuron, they are not immunoreactive with antibodies to sustentacular cells. The cells are stained, however, by an anti-neuronal antibody, NEU-9. The second aspect of altered organization is the appearance of novel olfactory marker protein-positive structures in the olfactory mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)
Differentiation of Friend erythroleukemia cells induced by benzodiazepines.
Many benzodiazepines of the type whose receptors are found in peripheral tissues cause the differentiation of cultured Friend erythroleukemia cells. The maximal level of induction of hemoglobin synthesis is 10-80% of the cells, depending on the compound tested. The induction is concentration and time dependent, requiring micromolar amounts of the drugs and about 5 days of treatment for full expression. The time course is very similar to that observed for a well-studied inducer, dimethyl sulfoxide. The affinities of the agents for the peripheral-type benzodiazepine binding site are not correlated with their capacity to induce differentiation. Also, the biological effect is stereospecific since the (3S) stereoisomer Ro11 -6896 is at least an order of magnitude more potent than its (3R) enantiomer, Ro11 -6893. The benzodiazepine effect exhibits definite structure-activity relationships. A 1-methyl group is an absolute requirement, although this is not sufficient in itself. Hydroxyl and methoxyl groups at the 4' position enhance the biological activity, but 4'-chloro decreases it. Substitutions at the 2', 6', and 4 positions also decrease the biological activity, as does the lack of a 2-carbonyl group. These data suggest that the benzodiazepines act in a specific manner to induce the differentiation of Friend erythroleukemia cells.
Isolation and sequencing of two cerebellum-specific peptides.
By comparing the HPLC elution profiles of peptides isolated from different brain regions, two cerebellum-specific species have been identified. The peptides were isolated by sequential chromatography on reverse phase, followed by ion-pairing HPLC using alkane sulfates as pairing reagents. The sequences of both peptides have been determined by gasphase Edman degradation and carboxypeptidase Y analysis and were confirmed by synthesis. The larger peptide, termed cerebellin, is a hexadecamer of primary amino acid sequence NH(2)-Ser-Gly-Ser-Ala-Lys-Val-Ala-Phe-Ser-Ala-Ile-Arg-Ser-Thr-Asn-His- OH. The second peptide is a pentadecamer with a primary sequence identical to residues 2-16 of cerebellin, the nomenclature of which is des-Ser(1)-cerebellin. Subcellular fractionation of cerebellum followed by ion-pairing HPLC analysis shows that both cerebellin and des-Ser(1)-cerebellin are enriched between 2.5- and 4-fold in the P2 crude synaptosomal fraction, suggesting their sequestration in some subcellular particle in vivo.
Benzodiazepines that bind at peripheral sites inhibit cell proliferation.
[3H]Ro5-4864 binds to mouse thymoma cells in a specific, saturable, and reversible manner. Scatchard analysis shows a single class of binding sites with a Kd of 4.4 nM and a Bmax of 477 fmol per 10(6) cells. This benzodiazepine binding site is of the peripheral type, based on the relative potencies of Ro5-4864 and clonazepam in competing for [3H]diazepam binding. Fifteen benzodiazepines that bind to this site with affinities ranging from 6 nM to 1 microM also reversibly inhibit the proliferation of thymoma cells in culture at the micromolar dose range. There is a strong positive correlation (r = 0.85) between the binding constants of these compounds for the peripheral-type sites and their ED50 in inhibiting the uptake of [3H]thymidine into the cells. These sites may be involved in the regulation of thymoma cell proliferation.
The promotion of mitosis in cultured thymic lymphocytes by acetylcholine and catecholamines.
Thymic lymphoblasts possess beta-adrenergic, dopaminergic and nicotinic receptors. When activated by high concentrations of adrenaline, isoprenaline, dopamine and acetylcholine, magnesium-dependent events are initiated, which culminate in mitosis. These events can be blocked by testosterone. The cells also possess muscarinic and alpha-adrenergic receptors which respond to low concentrations of acetylcholine, carbamylcholine and noradrenaline. In these cases calcium-dependent, oestradiol-blockable mechanisms are triggered which eventually lead to cell division.
Monoclonal antibodies to the rat olfactory sustentacular cell.
Immunization of BALB/c mice with rat olfactory epithelium permitted the production of a number of monoclonal antibodies directed towards cell types within the tissue. One of these antibodies, designated SUS-1, reacts specifically with cells located at the luminal surface of the olfactory neuroepithelium which possess a characteristic wine-glass body and a basal process which projects to the basement membrane. Thus these cells have the topographical and morphological properties of olfactory sustentacular cells. The SUS-1-positive cells are not stained by an antibody to the olfactory marker protein and neither does the SUS-1 antibody react with mature olfactory neurons in double labeling experiments. Upon unilateral olfactory bulbectomy there is a marked reduction in olfactory marker protein positive cells while there is no apparent diminution in the labeling of SUS-1 cells. These data show conclusively that the mature olfactory neurons and the SUS-1-positive cells are distinct from one another. SUS-1 staining is absent in all tissues examined except the olfactory epithelium. However, small discrete groups of cells in the lamina propria mucosae (often referred to as the submucosa) do stain in addition to the luminal sustentacular cell layer. The possibility is considered that the mature sustentacular cells may be related to these latter cells.