Mei Satake

Lipid composition of nerve cell perikarya

Abstract (1) The results are reported of a lipid analysis of nerve cell perikarya of pig brain stem with standard extraction procedures and analytical methods. (2) The lipid content per nerve cell perikaryon having a mean cell volume of 70 · 10−12 1 was 6,060 · 10−12 g. The lipid/protein ratio was 0.47. (3) Phospholipids and cholesterol were the major lipids, each occupying 87 and 9%, respectively, of the total lipid in the nerve cell perikaryon. Of the phospholipid components, phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine constituted 47, 28 and 9% of the total lipids respectively. (4) Glycolipids other than gangliosides were not detected colorimetrically. Sphingomyelin was negligible in its amount. (5) Fatty acids ranging fromC16:0toC22:6 were found in the phosphoglyceride fraction of the nerve cell perikaryon. The major fatty acids wereC16:0,C18:0andC18:1 in the phosphatidyl choline,C18:0andC22:6 in the phosphatidyl ethanolamine, andC18:0andC20:4 in the phosphatidyl serine. (6) With regard to the qualitative and quantitative order of the lipid components including the fatty acid, the nerve cell perikarya were opposite to the white matter, with the gray matter coming between the two.

Phosphorylation of neurofilament proteins and localization of axonal swellings in motor neuron disease

Seven lumbosacral spinal cords with motor neuron disease were examined immunocytochemically with monoclonal antibodies (MAb) directed against neurofilament proteins (NFP). Each of the 5 MAbs used in this study was monospecific to one of the triplet of NFP. Two of them were specific to the highly phosphorylated form of high molecular weight peptide of NFP (NFP-H). All 5 MAbs stained all axonal swellings examined. In 2 spinal cords examined, some axonal swellings were found in the anterolateral funiculus and some of these were as far as 1000 microns from the grey matter. This localization of axonal swellings suggests that a high degree of phosphorylation of NFP is not the cause of accumulation of NFP in axonal swellings in motor neuron disease.

Potential-dependent effects of sea anemone toxins and scorpion venom on crayfish giant axon

Effects of two kinds of sea anemone toxin (Parasicyonis actinostoloides andAnemonia sulcata) and scorpion venom (Leiurus quinquestriatus) on crayfish giant axons were examined electrophysiologically. All toxins acted on the axon in a similar manner to prolong the falling phase of the action potential. In all cases the development of toxicity was reduced when the nerve membrane was depolarized by a current injection. However, the ranges of membrane potential where the significant reduction in toxicity took place were different for each toxin. The action of Parasicyonis toxin was also suppressed by depolarization resulting from treatment of the axon with a neurotoxic alkaloid, veratridine. The mechanism of the potential-dependent toxin action is discussed with reference to the present data and relevant works by other investigators.

Blockade of [3H]lysine-tetrodotoxin binding to sodium channel proteins by conotoxin GIII.

Conotoxin GIII from Conus geographus inhibited the binding of [3H]lysine-tetrodotoxin (4 nM) to electroplax membranes from Electrophorus electricus and to the rat brain P2 fraction with IC50 values of 13 nM and 7.9 microM, respectively. This inhibition observed with electroplax membranes was irreversible. These and physiological findings (Life Sci., 21 (1977) 1759-1770 suggest that conotoxin GIII inhibits Na channel activation by its interaction with the tetrodotoxin binding site of the Na channel. The differences in structures related to the activation of Na channels between the eel electroplax and the rat brain are indicated.

Processing of neurofilament proteins from perikaryal to axonal type

In previous studies, neuronal cell bodies, excised by hand from bovine spinal ganglia, were analyzed and heterogeneous intermediate and high molecular weight neurofilament proteins that differed in electrophoretic mobility from their axonal counterparts were demonstrated (1, 2). In the present experiment, intermediate and high molecular weight neurofilament proteins of the axonal type were treated with alkaline phosphatase, and neurofilament proteins enriched in perikaryal type proteins were labeled with32P. Results showed that neurofilament proteins were phosphorylated after their translation, in the perikarya and the proximal portion of the axon, and suggested that phosphorylation was responsible for the differences between axonal and perikaryal neurofilament proteins.

Stimulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase with brain gangliosides

The holoenzyme of cAMP-dependent protein kinase (cAMP-kinase) partially purified from the particulate fraction of rat brain was stimulated by gangliosides. Among various gangliosides tested, GM1 was most potent, giving Ka value of 19.5 microM. The maximal activation of the kinase was obtained with 100 microM GM1 using kemptide as substrate. Gangliosides inhibited the kinase activity of the catalytic subunit of cAMP-kinase. Of various substrates tested, the ganglioside-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I and myelin basic protein, but not histone H1 and casein. The molecular mechanisms of the stimulatory effect of gangliosides were investigated. The kinase activated with GM1 was inhibited by the addition of PKItide, a specific inhibitor for cAMP-kinase. However, GM1 did not dissociate the holoenzyme into the catalytic and regulatory subunits and did not interfere with the binding ability of cAMP to the holoenzyme. These results suggest that the gangliosides can directly activate cAMP-kinase in a different manner from cAMP.

Photolabile μ-conotoxins with a chromogenic phenyldiazirine: A novel probe for muscle-type sodium channels

Three photoreactive derivatives of μ‐conotoxin G IIIA have been prepared as photoaffinity labeling reagents for muscle‐type sodium channels. The reagents competitively inhibited the binding of saxitoxin to the eel sodium channel with K i values of 11–18 nM. The introduced chromogenic phenyldiazirine group on the toxin was photolyzed efficiently, and spectroscopic properties of the reagents demonstrated that irradiation and detection can be performed in a spectral region where the absorptions due to most of biological macromolecules are negligible.

Acidic sphingoglycolipids in invertebrate nervous tissue: Presence of several kinds of sphingophosphonoglycolipids in the nervous tissue of Aplysia kurodai, a sea gastropod

Several hitherto unknown sphingophosphonoglycolipids were identified in the lipid extract from the nervous tissue, nerve fibers and ganglia of Aplysia kurodai, which has only a small amount of gangliosides, if any. The most abundant sphingophosphonoglycolipids of the nerve fiber (FGL-VII) and ganglion (GGL-V) are ceramide bis(2-aminoethylphosphono)-pentaosides. Their oligosaccharide moieties consist of 1 mol each of glucose, 3-O-methylgalactose and galactosamine and 2 mol of galactose. Another main glycolipid (FGL-II) may be ceramide mono(2-aminoethylphosphono)-pentaoside which contains 1 mol each of glucose, galactosamine and fucose and 2 mol of galactose as its oligosaccharide moiety.

Studies on the processing of 45-S RNA in rat liver nucleolus with specific reference to 29.5-S RNA

Abstract Nucleolar RNAs prepared from rat liver labelled with [ 14 C]- or [ 3 H]orotic acid with cold and hot sodium dodecyl sulphate-phenol extraction, were analysed by means of polyacrylamide gel electrophoresis and the following results have been obtained. 1. 1. In nucleolar RNAs which are prepared with cold sodium dodecyl sulphatephenol extraction not only from normal, but also from thioacetamide-treated and regenerating rat livers, 29.5-S RNA is found in addition to 45-, 37-, 36-, 32-, 28-, 21.5- and 18-S RNAs, whereas in those with hot sodium dodecyl sulphate-phenol extracttion, 29.5-S RNA is not observed in all cases. Nucleolar 28-S RNA and cytoplasmic 28-S RNA of rat liver co-migrate in acrylamide gel electrophoresis and do not change their electrophoretic mobilities with hot sodium dodecyl sulphate-phenol extraction or heat treatment, indicating that the presence of 29.5-S RNA is not due to the conformational change of 28-S RNA. The 37-S minor component is very unstable in the case of normal rat liver, but stable in the case of thioacetamide-treated rat liver. 29.5-S and 37-S RNAs are thought to be newly found RNAs using the cold sodium dodecyl sulphate-phenol extraction method. 2. 2. Taking the kinetics of labelling, the results of actinomycin D chase experiment and the molecular weights of the nucleolar RNA components together, may indicate that 29.5-S RNA is the metabolic intermediate between 32-S and 28-S RNAs, and 37-S RNA is suggested to be the direct precursor of 36-S RNA. The following scheme for the processing of 45-S RNA in rat liver nucleoli is proposed:

Conotoxin GIIIA: selective inhibition of 22Na influx via voltage-dependent Na channels in adrenal medullary cells

SummaryConotoxin GIIIA and GIIIB from the marine snail Conus geographus have been reported to inhibit voltage-dependent Na channels in skeletal muscle and postganglionic sympathetic neuron, but have no effect on Na channels in brain, giant axon and heart. In eel electroplax, conotoxins were also shown to share the common binding sites with saxitoxin (see review Gray et al. 1988).In bovine adrenal medullary cells, conotoxin GIIIA inhibited veratridine-induced influx of 22Na, 45Ca and secretion of catecholamines with an IC50 of 6 μmol/l, while saxitoxin suppressed veratridine-induced responses with an IC50 of 6.3 nmol/l. [3H]Saxitoxin binding to the cells was inhibited by unlabeled saxitoxin with an IC50 of 5.1 nmol/l, but was slightly reduced by 10 μmol/l conotoxin GIIIA. Conotoxin GIIIA, at 10 μmol/l, did not alter carbachol-induced influx of 22Na, 45Ca and secretion of catecholamines as well as high K-induced 45Ca influx and catecholamine secretion.These results indicate that conotoxin GIIIA, at concentrations 950 fold higher than saxitoxin, inhibits Na influx via voltage-dependent Na channels, but has no effect on the nicotinic receptor-ion channel complex or the voltage-dependent Ca channels. Conotoxin GIIIA seems to bind at the sites which are distinct from saxitoxin, but are functionally linked to the voltage-dependent Na channels. Conotoxins may be useful for the classification of Na channels in excitable cell membranes.