Regulation fo protein kinase C activity by various lipids

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracelluular communication.Abbreviations used (PK-C) Protein kinase C - (cAMP-PK) cAMP dependent protein kinase - (DAG) diacylglycerol - (PtdSer) phosphatidylserine - (InsP ₃) inositol 1,4,5-trisphosphate - (PtdIns 4,5-P₂) inositol 4,5 bisphosphate - (FFA) free fatty acid - (MBP) myelin basic protein - (ATP) adenosine triphosphate - (GTP) guanine triphosphate - (TPA) 12-tetradecanoylphorbol-13-acetate - (EGF) epidermal growth factor - (PDGF) platelet derived growth factor - (NeuNAc) and N-acetylneuraminic acid     

Ganglioside GD3: structure,cellular distribution,and possible function

Summary Insight on the function of gangliosides. can emerge from knowledge of their cellular distribution. In this paper we review the structure of ganglioside G_D3 and recent information on its cellular distribution. G_D3 appears to be enriched in a variety of neural cell types including: reactive glia, gliomas, undifferentiated neurons, Muller glia, and oligodendroglia. Because each of these cell types share an enhanced permeability to ions and metabolites or possess properties associated with enhanced permeability, we suggest that G_D3 is associated with enhanced membrane permeability. A possible function for G_D3 in membrane permeability has implications for other cellular events such as metabolism, growth and interactions.     

Confirmation of the Cholinergic Specificity of the Chol-1 Gangliosides in Mammalian Brain Using Affinity-Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus

An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cholinergic-specific immune lysis of mammalian brain synaptosomes and recognizes a group of minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyltransferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Analysis of glycosphingolipid-derived oligosaccharides by high pH anion exchange chromatography

As an adjunct to existing thin layer and column chromatographic methods for the identification of glycolipids a method that utilizes the high pH anion chromatographic (HPAEC) analysis of the oligosaccharides released from the glycolipids by endoglycoceramidase has been developed. Using a Dionex Carbo Pak PA1 column and elution with a linear gradient of sodium acetate in 0.2M NaOH, the elution times of eight neutral and fourteen acidic oligosaccharides derived from glycolipids were determined. Under these conditions the neutral oligosaccharides were well separated from each other but some of the acidic oligosaccharides had overlapping elution times. The ganglioside-derived oligosaccharides could be further identified by treating them with sialidase or by mild acid hydrolysis and reanalysing the products by HPAEC. The method was applied to the analysis of mixed bovine brain gangliosides. The procedure provides an additional approach for the initial identification of glycolipids by analysing the component oligosaccharides rather than the intact glycolipids.     

Functional relationship between ammonia and gangliosides in brain

The functional significance of ammonia production in brain under physiological or pathological conditions is not clearly known. NH₄ ⁺ stimulates Na⁺, K⁺ activated ATPase causing stabilization of neuronal membranes of which gangliosides are major structural components. Moreover ammonia is known to inhibit lysosomal enzymes which include enzymes degrading gangliosides. Gangliosides have been shown to stimulate neuritogenesis in neuronal cultures and prevent the damage of the neurons from glutamate toxicity particularly in areas of brain ischemia. Hyperammonemia without any behavioural changes was induced in experimental rats by intraperitoneal administration of either a single dose (0.8 mmol/100 g wt.) or by six hourly doses (0.6 mmol/100 g wt.) of ammonium acetate. An increase in the content of gangliosides along with a rise in the content of GD_1A and GD_1B without any change in -galactosidase and N-acetylhexosaminidase was observed in cerebral cortex, cerebellum, and brain stem, following the administration of single dose of ammonium acetate. Gangliosides, after extraction from the different brain regions, were estimated by the thiobarbituric acid method and expressed in terms of sialic acid. Individual gangliosides were separated and estimated by thin layer chromatography using resorcinol as the staining agent. These results suggest that ammonia production in the neuronal pathways in brain either as a result of repeated stimulation under physiological conditions or as a result of focal ischemia or injury, may likewise cause an increase in the content of gangliosides which may help in neuritic growth (physiological conditions facilitating synaptic plasticity) and may exert a protective effect on the neurons in the ischemic area against glutamate toxicity.Former Professor of Biochemistry, OMC, Hyderabad.     

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, G_M3(Neu5Ac), G_M3(Neu5Gc) and G_M1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was G_M3(Neu5Ac) followed by moderately expressed G_M3(Neu5Gc) and G_M1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-G_M3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle. Abbreviations: BSA, bovine serum albumin; DAPI, 4, 6-diamidine-2-phenylindole-dihydrochloride; DTAF, dichlorotriazinylamino-fluorescein; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycolylneuraminic acid [53]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [54] and the nomenclature of Svennerholm [55]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gal1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₃Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₅Cer, GalNAc1-3GalNAc1-3GAl1-4Gal1-4Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer.     

Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase

Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid-bound sialic acid exceeds that of the protein-bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.