Stearylamine permeabilizes the lysosomal membrane to cystine and sialic acid

Cystine efflux from isolated rat liver lysosomes was enhanced by concentrations of stearylamine that were above the critical micellar concentration. Lysosomal latency, pH, and activity of the proton-translocating ATPase were largely unaffected under controlled experimental conditions. Loss of lysosomal latency was observed at higher stearylamine to protein ratios consistent with a detergent-like mechanism of action. Partially purified cultured fibroblast lysosomes with either defective cystine or sialic acid transport lost their stored material upon exposure to stearylamine. Concentrations of stearylamine which were effective for lysosomal efflux were highly toxic for cultured fibroblasts, thus limiting its use. Under specific conditions, stearylamine apparently selectively permeabilizes the lysosomal membrane. A similar acting, but less toxic agent may be of use in the treatment of lysosomal transport disorders.     

Erythrocyte membrane bound and plasma sialic acid during aging

Sialic acid, a nine-carbon sugar, is an acetylated derivative of neuraminic acid predominantly found in vertebrates, a few higher invertebrates, and certain types of bacteria. Red blood cells (RBCs) have a net negative surface charge and this bulk charge is due to ionized sialic acid. Decreased surface charge and sialic acid content have been reported in older erythrocytes, and it is postulated that the decreased electro-negativity may be related to cell senescence. In the present study we report the RBC and plasma sialic acid content during aging in rats. Our results show a significant decrease in RBC sialic acid content and increase in plasma sialic acid as a function of rat aging. The decreased sialic acid in erythrocyte membrane with increasing rat age presents a good biomarker of the aging process. The elevated plasma sialic acid may be a manifestation of several factors including increased expression of acute phase proteins and increased damage to various organs.     

Reduced levels of sialic acid in the plasma membrane during hepatocellular proliferation

When rats were infused with a solution containing triiodothyronine, amino acids, glucagon and heparin (solution A) the hepatocytes increased DNA synthesis and decreased plasma membrane sialic acid. In order to study whether the reduced levels of sialic acid in the plasma membrane were associated with hepatocyte proliferation, different mixtures of three components of solution A were infused into rats and the DNA synthetic activity as well as the sialic acid content measured. Results reported here show a correlation between DNA synthetic activity and sialic acid reduction suggesting that the decrease in the plasma membrane sialic acid can be a pre-replicative step associated to cell proliferation.     

The linkage of sialic acid in the Ehrlich ascites-carcinoma cell surface membrane

1. The sialic acid content of fresh and fixed Ehrlich ascites cells was determined by incubation with neuraminidase and analysis of the supernatants. 2. The content of sialic acid was also determined on ultrasonically disrupted cells either with or without prior neuraminidase treatment, and the location of sialic acid in the cell is discussed. 3. The sialic acids, cleaved from cells by neuraminidase, were identified chromatographically. 4. Proteolytic enzymes were used to isolate from cells a mucopeptide containing sialic acid and galactosamine in almost equimolar proportions. 5. The nature of the carbohydrate-amino acid bond in the muco-peptide was investigated by alkaline hydrolysis. 6. A suggestion is made about the particular amino acids involved in the sugar-peptide bond.     

Changes in sialic acid content of the plasma membrane in hepatocellular proliferation

The content of sialic acid bound to the sinusoidal region of plasma membrane during the prereplicative phase after the intravenous injection of a solution containing triiodothyronine, amino acids, glucagon and heparin (T.A.G.H. solution) has been measured. The results obtained show that an important decrease in sialic acid content is produced as it occurs in the hepatic cells of hepatectomized animals. In order to know if sialidase activity is involved in the decrease of sialic acid content during liver regeneration, the activity of sinusoidal plasma membrane sialidases during the prereplicative phase after the partial hepatectomy has been studied. No modifications of sialidase activity were detected during this period of time indicating that this decrease in sialic acid content has to be produced by other mechanisms such as diminution in the synthesis of precursor molecules. On the other hand due to the importance of Ca2+-calmodulin complexes in the activation of the hepatic cell proliferation the possible implication of this complex on the loss of sialic acid, observing the effect of trifluoperazine (inhibitor of Ca2+-calmodulin complexes) during the prereplicative phase of liver regeneration has been studied. The results show a delay in the decrease of the amount of sugar studied from 10 to 12 hours compared to the results obtained with the hepatectomized rats that have not received trifluoperazine.     

Release of sialic acid alters the stability of the membrane potential in cardiac muscle

Intracellular recording techniques and neuraminidase, an enzyme that specifically catalyzes the hydrolysis of sialic acid's glycosidic linkage in glycoproteins and glycolipids, were employed to investigate the role of sialic acid residues in maintaining a stabilized resting potential or rhythmic electrical activity in embryonic chick cardiac muscle. Free sialic acid was quantified by a fluorometric assay. Release of more than 25% of the sarcolemma-bound sialic acid from spheroidal aggregates of cultured heart cells resulted in a) depolarizing fluctuations in the membrane potential, b) initiation of spontaneous firing in the presence of tetrodotoxin, c) arrhythmic spontaneous activity, d) depolarization of the maximum diastolic potential, and e) a significant reduction in the plateau and duration of the action potential. Control experiments demonstrated that these effects were not caused by phospholipase contamination of the enzyme or by the sialic acid released during hydrolysis.     

Association of sialic acid with microsomal membrane structures in rat liver

The amount of sialic acid on phospholipid basis increases from rough, through smooth II and smooth I microsomes, to Golgi membranes, all of them free from most of the adsorbed and luminal protein. The incorporation rate of glucosamine-³H into sialic acid also follows a similar order. Deoxycholate removes phospholipid and sialic acid to an identical extent, and a significant part of the latter remains after trypsin and neuraminidase treatment. The sialic acid/phospholipid ratio decreases in phenobarbital-induced smooth but not in rough membranes, while the incorporation rate of glycosamine-³H into sialic acid decreases in both subfractions.     

Synthesis of sialic acid derivatives as ligands for the myelin-associated glycoprotein (MAG)

The trisaccharide substructure 13 of the ganglioside GQ1balpha shows a remarkable affinity for the myelin-associated glycoprotein (MAG). In the search for structurally simplified and pharmacokinetically improved mimics of 13, sialosides with modifications at the reducing and non-reducing end were synthesized. The biological evaluation of mimics 12a-o was performed in a competitive target-based assay. It was found that the relative inhibitory potency (rIP) of antagonist 12h was enhanced by more than 1000-fold in comparison to the reference trisaccharide 13, despite the former having a much simpler structure. In addition, the sialic acid derivatives, for example, 12h, have clearly improved pharmacokinetic properties due to the presence of aromatic moieties, a lower molecular weight, and a reduced number of polar hydroxy functions compared to the reference compound 13.     

Demonstration of sialic acid groups in the glomerular basement membrane of the rat with phosphotungstic acid at low pH

Summary This paper reports an unrecognized aspect of phosphotungstic acid staining at low pH. It provides an on-section staining method in which sialic acid-containing molecules can be demonstrated in the laminae rarae of the rat glomerular basement membrane. The staining in the basement membrane became negative after perfusion with the following cations: protamine sulphate, hexadimethrine, Alcian Blue, Ruthenium Red and Toluidine Blue. Blocking ws not achieved with Alcian Blue at about pH 1. The staining was also abolished after mild methylation and demethylation restored the contrast. This is suggestive of the involvement of carboxyl groups. Prior digestion with pronase, trypsin and neuraminidase rendered the laminae rarae negative, whereas hyaluronidase, chondroitinase ABC and crude heparinase were without effect. This indicates that sialic acid groups are detected by this method and that heparan sulphate does not interfere. The staining of the epithelial plasma membrane, also carrying sialic acid groups, remained positive after neuraminidase treatment. It is presumed that this method can be applied successfully for detecting changes in the sialic acid content of the laminae rarae in rat glomerular basement membranes under normal and pathological conditions.     

Removal or oxidation of surface membrane sialic acid inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis

Polymorphonuclear leukocyte (PMN) surface membrane glycoproteins are probably involved in the phenomenon of stimulus-response coupling. Consequently, we examined the effects of either removal or oxidation of surface membrane-associated sialic acid residues on some responses of human PMN to chemotactic factors. Treatment of human PMN with either neuraminidase or sodium metaperiodate did not affect the ability of these cells to migrate randomly, but did inhibit their ability to respond chemotactically to the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). Treated PMN responded normally, however, to the complement-derived peptide C5a, and to the lipoxygenase product leukotriene B4. Enzymatic removal or oxidation of membrane sialic acid residues did not affect either FMLP-induced PMN degranulation or FMLP-induced generation by PMN of superoxide anion radicals. Removal of sialic acid did not significantly alter specific binding of [3H]FMLP to its receptor(s) on the PMN membrane. These findings indicate that sialic acid residues on the PMN surface membrane play an important role in modulating PMN responses to FMLP.     

Abscisic acid increases membrane permeability by interacting with phosphatidylethanolamine

Abscisic acid is shown to enhance the permeability of crude egg lecithin and asolectin bilayers to water, urea and erythritol although it exhibits no effect on pure synthetic (phosphatidylethanolamine-free) dimyristoylphosphatidylcholine bilayers. Addition of dipalmitoylphosphatidylethanolamine to dimyristoylphosphatidylcholine bilayers at 10 or 20 membrane mole percent makes the membrane permeability responsive to abscisic acid. An abscisic acid-phosphatidylethanolamine interaction is also described for liposome aggregation. Both abscisic acid-induced permeability and aggregation changes are pH dependent with the undissociated form of the hormone exhibiting a greater effect than the dissociated, charged form. Enhancement of erythritol permeability is greater with the physiologically active cis-trans ABA isomer than with the inactive trans-trans isomer.     

Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane

Summary Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane was studied. The transport properties, such as pH-dependence and effects of reagents reacting with amino-groups, were similar to those of anions like Cl^– through the human erythrocyte membrane. The rate of transport of anions through rat erythrocyte membranes is higher than through those of other mammals, such as guinea pig and bovine erythrocyte membranes. This relatively high rate of transport makes the rat erythrocyte membrane suitable for use in comparative studies on the transports of slowly penetrating substances, such as organic anions. The transport velocities of benzenesulfonic acid derivatives were compared with their physico-chemical properties. It was shown that the hydrophobicity has no effect on the transport, but the electronic property has a significant effect: the transport rate is mainly dependent on thee ^– donor capacities. This feature is the inverse to the well-known inhibitory effect of these derivatives on other anion transport: the inhibition is mainly dependent on thee ^– acceptor capacities. It is suggested that the transport is regulated by the binding capacity of anions to the transport site.     

Affinity of mouse immunoglobulin G subclasses for sialic acid derivatives immobilized on dextran-coated supports

High-performance liquid affinity chromatography is a powerful method for the purification of biological compounds owing to its specificity, rapidity and high resolution. In our laboratory, we develop chromatographic supports based on porous silica beads. However, in order to minimize non-specific interactions between the inorganic surface and proteins in aqueous solution, the silica beads are coated with modified dextran. As previously reported, many affinity ligands can be covalently grafted onto dextran-coated silica. In this study, N-acetylneuramic acid, which belongs to the sialic acid family and is present in immunoglobulin G (IgG) epitopes, is used as an active ligand. The interactions of this affinity support and IgG subclasses are analyzed. This immobilized ligand enables purification of IgG3 antibodies.     

Arachidonic acid induces specific membrane permeability increase in heart mitochondria

Micromolar concentrations of arachidonic acid cause in Ca2+ loaded heart mitochondria matrix swelling and Ca2+ release. These effects appear to be unrelated to the classical membrane permeability transition (MPT), as they are CsA insensitive, membrane potential independent and can also be activated by Sr2+. Atractyloside potentiated and ATP inhibited the arachidonic acid induced swelling. These observations suggest that the ATP/ADP translocator (ANT) may be involved in the AA induced, CsA insensitive membrane permeability increase. Under the same experimental conditions used for heart mitochondria, arachidonic acid induced the classical CsA sensitive, ADP inhibitable MPT in liver mitochondria.     

Mass spectra of acetylated derivatives of sialic acids

The fragmentation pattern in electron-impact mass spectrometry has been established for the peracetylated methyl ester methyl glycoside derivative of N-acetylneuraminic acid. The resulting, data allow the interpretation of the mass spectrum of the corresponding derivative of a new sialic acid isolated from the starfish Distolasterias nipon which is shown to be 8-O-methyl-N-acetylneuraminic acid.     

Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.