Neural cadherin: role in selective cell-cell adhesion.

Cadherins are a family of Ca2+-dependent intercellular adhesion molecules. Complementary DNAs encoding mouse neural cadherin (N-cadherin) were cloned, and the cell binding specificity of this molecule was examined. Mouse N-cadherin shows 92 percent similarity in amino acid sequence to the chicken homolog, while it shows 49 percent and 43 percent similarity to epithelial cadherin and to placental cadherin of the same species, respectively. In cell binding assays, mouse N-cadherin did not cross-react with other mouse cadherins, but it did cross-react with chicken N-cadherin. The results indicate that each cadherin type confers distinct adhesive specificities on different cells, and also that the specificity of N-cadherin is conserved between mammalian and avian cells.

In situ elemental analysis and visualization in cryofixed nervous tissues. X-ray microanalytical investigations of embryological and mature brain, inner ear, photoreceptors, muscle and muscle spindles. Comparison of preparation methods for analysis and visualization at cellular and subcellular levels.

For meaningful X-ray microanalysis (XRMA) in biology and medicine, the development of preparative and quantitative methods has been necessary. The methods need to preserve close to in vivo distribution of diffusible ions with at the same time reasonable morphological preservation of the tissue. Analyses at low and middle microanalytical resolution are useful at the initial stages of an investigation or when data from large populations of samples have to be acquired. Cryomicrotomy, which makes it possible for the single cells within semi-thin and thick cryosections examined by X-ray microanalysis to be further characterized histochemically (enzyme and substrate content), has been adopted for several pathophysiological studies. The method is particularly suitable for the analysis of complex morphological tissues with many cell types as in the brain or sensory organs of the internal ear. For microanalysis at the subcellular level, we developed a preparative procedure based on the frozen fixed preparation which is freeze-dried in vacuo at -80 degrees C and then at the same temperature, without breaking the vacuum, impregnated with a low-temperature Lowicryl-type resin. The resin is polymerized by u.v. light. This method prevents redistribution of the ions in the tissue and retains the antigenicity of the tissue. A considerable number of cells can be analysed simultaneously and the elemental composition in different cell compartments can be compared due to the similar analytical conditions within the section. An alternative to thin plastic sections of freeze-dried material is thin cryosections cut at -150 degrees C and analysed at low temperatures. Although some methodological problems still exist in preparation of cryosections, this type of section is potentially the most useful in analysis of diffusible ions, especially calcium which in most biological systems is present in very low concentrations. New preparative techniques for XRMA brought severe problems in visualization of the specimens prepared by cryomethods. Charging, low contrast, mass loss and contamination, which are often negligible in conventional electron microscopy, have still to be solved in XRMA of cryoprepared specimens. However, the methods of semi-thin and thick cryosectioning and low-temperature embedding were successfully used for analysis of cells and organelles and for the study of fluids in restricted biological spaces such as the inner ear, muscle spindles and ventricles of the brain in rats. Accordingly, examinations which were impossible by micropuncture and ion selective techniques could be carried out by XRMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.

An immunological study on chromogranin A and B in human endocrine and nervous tissues.

Antisera were raised against synthetic peptides derived from the primary amino acid sequence of human chromogranin B. These antisera recognized in one- and two-dimensional immunoblotting a component previously designated as chromogranin B. In human chromaffin granules, the major endogenous processing product of chromogranin B is formed by proteolytic cleavage of the protein near the C-terminus. Immunohistochemical localizations were obtained with antisera against human chromogranins A and B and against a synthetic peptide corresponding to the B sequence. In human tissues, chromogranin B is co-stored with chromogranin A in the adrenal medulla, the anterior pituitary, parafollicular cells of the thyroid, in some cells of the endocrine pancreas and in some enterochromaffin cells, whereas only chromogranin A is found in the parathyroid gland and enterochromaffin cells of the gastric corpus mucosa. In the nervous system, no immunostaining was observed for chromogranin A and only a weak one for chromogranin B in some cells of the spinal cord. However, the Purkinje cells of the cerebellum were strongly positive for chromogranin B.

Possible involvement of polysialogangliosides in nerve sprouting and cell contact formation: an ultracytochemical in vitro study.

In Cichlid fish (Oreochromis mossambicus) primary cell cultures from whole brain and optic tectum, the differentiation-dependent distribution of polysialogangliosides on the outer cell surface has been followed on an ultrastructural level. For this, a two-step labeling technique with the monoclonal mouse antibody Q211, recognizing a polysialoganglioside-associated epitope, followed by a secondary IgM antibody, coupled to colloidal gold sols as an electron-dense marker, has been used. The gold grains are not uniformly distributed over the whole cell surface, but rather are clearly arranged clusters. In cells from freshly hatched larvae, both cell bodies and nerve fibers strongly exhibit the polysialoganglioside epitope on their surface. With progressing development, neuronal cell labeling is more and more restricted to nerve fibers and especially to cellular adhesion zones, including synaptic terminals, thus suggesting a functional involvement of polysialogangliosides in nerve sprouting and initiation of both cell-to-extracellular matrix and cell-to-cell contacts.

Axolinin localization in the nervous tissue of squid revealed by monoclonal antibodies specific for axolinin: characterization of monoclonal antibodies against axolinin.

Monoclonal antibodies against squid axolinin (the 260-kD glycoprotein in squid axoplasm) have been produced using spleen cells immunized with purified axolinin in vivo and in vitro. The produced antibodies can be categorized into two groups according to the nature of their antigenic site. The monoclonal antibodies belonging to group I recognize the protein backbone of axolinin, while those belonging to group II bind to the sugar chains of axolinin. Group II was further divided into two subgroups IIA and IIB; the subgroup IIA antibodies recognize sugar chains to which concanavalin A binds, while the IIB antibodies bind to sugar chains lacking the lectin-binding sites. Only the group I antibodies can be used as specific probes for axolinin since the sugar chains recognized by the group II antibodies are present on other squid glycoproteins in addition to axolinin. It follows from our results that polyclonal antisera against axolinin cannot generally be expected to be specific for this protein.

[Localization and functional role of gangliosides from nerve tissue of vertebrates]. / Lokalizatsiia i funktsional'naia rol' gangliozidov nervnoi tkani pozvonochnykh.

Gangliosides in the animal organism are typical components of plasma membranes of nerve cells in which the concentration of polysialogangliosides is especially high. The high concentration of tri- and tetrasialoganyliosides and ganglioside GD1b is peculiar to primary cultures of nerve cells, whereas these gangliosides are practically not present in the culture of the transformed nerve cells which have lost their ability to sinaptogenesis, they are not found in cultures of oligodendro- and astroglia as well. The addition of exogenic gangliosides to nerve cells cultures stimulates the formation of processes in these cells and promotes their survivability. The neuritogenic and neuronotrophic effect of gangliosides, their participation in the processes of neurons' regeneration are shown in the in vivo experiments. Gangliosides are carriers of antigenic determinants typical of the cellular surface of neurons (or other cells) as well as of cells of different malignant tumours; typical carcinoembryonal antigenes are revealed among them. Such functions of gangliosides as participation in the processes of intercellular interaction, adhesion, pneuritogenic effect, possible participation to memorize one or another habit are, probably, interrelated and mutually conditioned. Recently the data, being in controversy with the notion that gangliosides are components of receptors of hormones and mediators, are obtained. Evidently, it ought to speak about the modulation of cell response by them on the action of these effectors.

Characterization and comparison of lipids in different squid nervous tissues.

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.

Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.

Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.

Studies on the neurobiology of vitamin E (alpha-tocopherol) and some other antioxidant systems in the rat.

To understand why nervous tissue should be particularly affected by severe deficiency of vitamin E, the distribution of vitamin E (alpha-tocopherol) and some other antioxidant systems was investigated. The concentration of vitamin E and the activities of glutathione peroxidase and superoxide dismutase were determined in different regions of the nervous system in male Wistar rats. The cerebral cortex had the highest, and the cerebellum the lowest concentration of alpha-tocopherol (P less than 0.02). Activity of glutathione peroxidase tended to show an inverse relationship to the alpha-tocopherol concentration, whereas superoxide dismutase activity was evenly distributed through the nervous tissue. Vitamin E concentrations were also determined in spinal cord, sciatic and tibial nerves and in epineurial, myelin and non-myelin fractions of the sciatic nerve. Uptake of intravenously injected tritiated alpha-tocopherol was studied after 6 h and found to be greater in brain and peripheral nerve than cervical and thoracic regions of the spinal cord. Uptake of tocopherol varied along the sciatic and tibial nerve, being greatest where the sciatic nerve divided into tibial, sural and common peroneal nerves. This corresponded to an area of increased vasculature which was visualized by an angiographic technique using barium sulphate.

Determination of chinoform in biological fluids and nervous tissues of the dog by gas chromatography-mass spectrometry.

A sensitive gas chromatographic-mass spectrometric method for the determination of 5-chloro-7-iodo-8-hydroxyquinoline (chinoform, clioquinol) in biological fluids and nervous tissues is described. Chinoform was converted into the pentafluorobenzyl ether, which was separated on a 10% Dexsil 300GC column and determined by the use of chinoform-d4 as an internal standard. The clean-up of chionoform in plasma and urine was efficiently achieved by extracting with benzene, while the drug in the tissue was pretreated successively by extraction with 12.5% v/v pyridine-benzene, separation on a Clin-Elut cartridge and adsorption on alumina. The quantitation limit of chinoform was 100 pg, and the recovery rates of chinoform added to plasma and tissue were 98% and 92%, respectively. The chinoform levels in biological fluids and tissues in dogs after prolonged administration of the drug at a dose of 400 mg/kg/day were measured by the proposed method. The plasma level and tissue distribution of chinoform are also discussed.

Messenger RNA from insect nervous tissue induces expression of neuronal acetylcholine receptors in Xenopus oocytes.

mRNA isolated from the nervous tissue of young insects microinjected in Xenopus oocytes induced the expression of alpha-toxin binding sites which were inserted into the surface membrane. Immunoprecipitation experiments revealed that the coded receptor polypeptides were processed to the authentic size, and ion flux studies demonstrated that functional nicotinic acetylcholine receptors were produced and inserted into the oocyte membrane.

Characterization of epitopes shared by alpha-melanocyte-stimulating hormone (alpha-MSH) and the 150-kD neurofilament protein (NF150): relationship to neurotrophic sequences.

Immunoblot techniques and immunohistochemistry have been used to investigate epitopes shared by alpha-melanocyte-stimulating hormone (alpha-MSH) and the 150-kD neurofilament protein (NF150). Three anti-alpha-MSH antisera (namely 31H2T, 4394, and M5) from a total of 12 sera were found to react with NF150. The three crossreacting sera are different in their binding properties to peptide fragments related to alpha-MSH, suggesting that at least two distinct epitopes are shared by NF150 and alpha-MSH. The only known sequence common to NF150 and alpha-MSH, the N-terminal Ac-S-Y residues, appears to be essential for binding of sera 31H2T and 4394. However, the binding of antiserum M5 involves other, currently unknown, similarities between NF150 and alpha-MSH. This is shown by the binding of M5 to peptides such as ACTH(4-10), which do not contain the N-terminal Ac-S-Y sequence. Binding of M5 to tobacco mosaic virus coat protein (TMV-coat protein), which is homologous with alpha-MSH and NF150 in its Ac-S-Y residues, was negligible. The peptide structures that are recognized by M5 have previously been shown to exert neurotrophic activity. The data are discussed in the light of the hypothesis that similarity between NF150 and alpha-MSH, as illustrated by binding to M5, may be significant in the neurotrophic activity of MSH-related peptides.

Peroxisomes in the nervous system of Aplysia californica: a cytochemical study.

We have studied the distribution of peroxisomes in the abdominal ganglion of Aplysia californica using electron microscopic cytochemical methods. Reaction product for catalase was observed in small ovoid or dumb-bell-shaped bodies in the perikarya of many of the neurons. The abundance of these catalase-reactive peroxisomes is considerably greater than is the case in vertebrate neurons. While the non-neuronal cells of the Aplysia abdominal ganglion do contain appreciable peroxisome populations, there were few peroxisomes in glial cytoplasm directly adjacent to the perikarya, again contrasting with vertebrate ganglia in which the satellite cells are a principal site of peroxisomes. Peroxisomes are present throughout the perikaryal cytoplasm. In the regions in which lipochrome granules abound, peroxisomes are frequently seen closely associated with these granules; glycogen is abundant nearby. The association of peroxisomes, lipochrome granules and glycogen is interesting in view of the propinquities of peroxisomes to lipid droplets and lipofuscin granules reported for non-neuronal vertebrate tissues, and in view of the growing evidence indicating that some of the roles of peroxisomes are in lipid metabolism and in gluconeogenesis. Some of the lipochrome granules themselves show reaction product in ganglia incubated to demonstrate catalase activity and some react in tissue incubated to demonstrate acid phosphatase activity. Such observations suggest that the enzymatic capacities of the lipochrome granules merit further studies, and that the granules may be of complex or heterogeneous nature.

Varicosity-associated antigens define neuropile subfields in the leech central nervous system.

A panel of twelve monoclonal antibodies raised against homogenates of leech nerve cords and four polyclonal antisera raised against purified neurotransmitters were used to label varicosities immunocytologically in the central neuropile of leech segmental ganglia. These immunoreactive varicosities occur in distinct patterns, some of which have a simple geometry. Three antibodies label immunoreactive varicosities distributed in a single dorsoventrally-oriented plane, two label varicosities distributed in lateral hemi-neuropiles (leaving void a central cephalocaudal passageway), and five label varicosities distributed throughout the neuropile. Six antibodies tested label varicosities across leech species, and five of these varicosity populations are distributed in patterns conserved across leech species. Immunocytologically-defined neuropile subfields do not correspond with previously identified histological and ultrastructural features of leech segmental ganglia. Analysis of immunocytologically-defined subfields is extended to include identification of sets of neurons which appear to project into these subfields, and to include an intracellular characterization of one of these neuron sets.

Phylogenetic survey of proteins related to synapsin I and biochemical analysis of four such proteins from fish brain.

A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.

The effects of valproate and phenytoin on the cAMP and cGMP levels in nervous tissue.

Earlier studies have demonstrated that valproic acid (VPA) and phenytoin (PHT) influence the excitability properties of crayfish axons through different mechanisms. PHT was found to antagonize the electrophysiologic effects of VPA. The purpose of the present study was to determine if the electrophysiologic effects of VPA and PHT are correlated with changes in the cellular levels of either cAMP or cGMP as these substances are known to influence membrane excitability. It was found that PHT (0.1 mM) has no effect on the levels of either cAMP or cGMP within crayfish neural tissue. VPA (4.0 mM) also has no effect on cAMP levels. However, it does significantly reduce the levels of cGMP. Pretreatment of neural tissue with PHT has been shown to eliminate the effects of VPA on membrane excitability. It was found that this pretreatment has no influence on VPA's ability to reduce cGMP levels. The effect of VPA on cGMP levels is observed in the absence of spontaneous activity. Therefore, it is concluded that the observed reduction in cGMP levels does not represent the modulation of cGMP levels that is known to accompany activity. Two experiments demonstrate that the 4-mV depolarization of membranes by VPA can not account for its effect on cGMP levels. In the first, pretreatment with PHT abolished the depolarizing effect on VPA but not its effect on cGMP. In the second, a concentration of ouabain which depolarizes crayfish neural tissue by 8-10 mV without producing spike activity had no effect on either cAMP or cGMP levels. These experiments effectively dissociate the electrophysiologic response to VPA and PHT from changes in cyclic nucleotide levels.

Insect cardioactive peptides. II. Neurohormonal control of heart activity by two cardioacceleratory peptides in the tobacco hawkmoth, Manduca sexta.

The physiological characteristics of two cardioacceleratory peptides (CAPs) were analysed in the tobacco hawkmoth, Manduca sexta, to determine if either CAP functioned as a cardioregulatory neurohormone. In vivo heart recordings from pharate and newly emerged adults revealed a dramatic increase in heart rate associated with wing-spreading behaviour. Bioassay of whole blood taken from wing-spreading (WS) animals indicated the presence of a stage-specific, blood-borne cardioacceleratory factor(s). Gel filtration of WS blood identified two cardioacceleratory factors which co-eluted with the two CAPs. A depletion of the ventral nerve cord levels of both CAPs was observed during WS behaviour. Measurements of blood CAP levels showed that the peak CAP titres were coincident with the initiation of WS behaviour. Experimental manipulations that delayed the onset of WS behaviour also prevented CAP release. High potassium incubation evoked the release of both CAPs in a calcium-dependent manner. In vivo injections of CAP1 or CAP2 caused a dose-dependent increase in heart rate. These results confirm the hypothesis that both CAPs function as cardioregulatory neurohormones during wing-spreading behaviour in Manduca sexta.