Quantitative analysis of brain gangliosides by high performance liquid chromatography of their perbenzoyl derivatives

This report describes a convenient, highly sensitive, and reproducible HPLC procedure for the quantitative analysis of gangliosides from brain tissues. The procedure involves the conversion of gangliosides to their perbenzoyl derivatives, isolation of the derivatives on a C18-reversed-phase cartridge, separation of the derivatives on a column (3-micron silica) maintained at an elevated temperature, and UV detection of the derivatives at 230 nm. The convenience of the procedure, its sensitivity, reproducibility, and application to the analysis of gangliosides from tissue sources make it the method of choice for ganglioside quantification in our laboratories. Three aspects of the procedure contribute to its convenience: reaction conditions that lead to single products, a convenient isolation procedure for the derivatives, and chromatographic conditions that provide resolution of the derivatives.     

Binding and activation of the first component of human complement on artificial matrices

Artificial sorbents that comprise macroporous glass covered by the copolymer of N-vinylpyrrolidone and N-substituted acrylamide have been synthesized. Aminoethanol is bound to acrylic acid residue in one sorbent (AE-glass), whereas the other sorbent involves immunoglobulin G with the hexamethylenediamine spacer (IgG-glass). C1q binds specifically to IgG-glass with Ka 4,07(+/- 0,32) X 10(7) M-1. Free energy of the C1q binding to IgG-glass is twice higher than that of its binding to monomeric IgG. This evidences that one C1q molecule associates with two IgG molecules of the sorbent. A weak nonspecific sorption of C1q to AE-glass was found. Both specific (on IgG-glass) and nonspecific (on AE-glass) sorption of the first component of complement activate the classical pathway in human serum as manifested in the consumption of the C4, C2, C3 and C5 components. IgG-glass was employed for C1q isolation from human serum by affinity chromatography, whereas unbound part of serum may be used as a reagent R1q. The yield of highly purified C1q after IgG-glass affinity chromatography and gel filtration on Sephacryl S-300 is 63,6%.     

In vitro and in vivo release of albumin using a biodegradable MPEG-PCL diblock copolymer as an in situ gel-forming carrier

An MPEG-PCL diblock copolymer was synthesized as an in situ gel carrier, and its phase transition behavior in aqueous solutions was examined. For comparison, aqueous solutions of Pluronic F-127, a widely used injectable gel-forming solution, were also studied. Both MPEG-PCL copolymer and Pluronic aqueous solutions were sols at room temperature. As the temperature was increased above room temperature, the diblock copolymer and Pluronic solutions underwent a sol-to-gel phase transition, which manifested as an increase in viscosity indicative of the formation of a gel. All of the copolymer solutions became gels at body temperature, although the gel viscosity increased with the increasing concentration of the MPEG-PCL diblock copolymer in the solution. In in vitro experiments, in which the gels were exposed to PBS, the MPEG-PCL gels maintained their structural integrity for more than 28 days, whereas the Pluronic gel disappeared within 2 days. The same results were observed when the polymer solutions were subcutaneously injected into rats. The MPEG-PCL gels maintained their structural integrity longer than 30 days, while the Pluronic gel could not be observed after 2 days. The ability of the gels as drug carriers was studied by measuring the release of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) from MPEG-PCL diblock copolymer gels in vitro as well as in vivo. In vitro, BSA release was sustained above 20 days, with a greater release at lower diblock copolymer concentration; by contrast, Pluronic gels exhibited almost complete release of BSA-FITC within 1 day. When the BSA-FITC-loaded diblock copolymer and Pluronic solutions were subcutaneously injected into rats, they immediately transformed into a gel. In vivo, sustained release of BSA-FITC over 30 days was observed from the MPEG-PCL gel, whereas BSA-FITC release from the Pluronic gel ceased within 3 days. Collectively, the present findings show that MPEG-PCL diblock copolymer solutions are thermo-responsive and maintain their structural integrity under physiological conditions, indicating that they are suitable for use as injectable drug carriers.     

Detection, determination, and metabolism in vitro of gangliosides in mammalian sympathetic ganglia

Abstract— Sympathetic ganglia of the rat and cat were examined for the occurrence and distribution of gangliosides. Each rat superior cervical ganglion contained 0.3 nmol of ganglioside-sialic acid. Extracts of cat superior cervical and nodose ganglia were chromatographed on silica gel thin-layer plates. The resulting patterns suggested that similar distributions of multiple forms of gangliosides occur in these two tissues, with the fast-moving gangliosides predominating. The metabolic activity of gangliosides was also investigated in rat superior cervical ganglia in vitro. Evidence was obtained that ¹⁴C from [U-¹⁴C]glucose, [U-¹⁴C]pyruvate, and [U-¹⁴C]glucosamine was incorporated into the gangliosides.     

Aminopropyl silica gel as a solid support for preparation of glycolipid immunoadsorbent and purification of antibodies.

Aminopropyl silica gel was prepared from porous silica gel and was used as a solid support for immunoadsorbent in the purification of anti-glycolipid antibodies. For neutral glycosphingolipids, a carboxyl function was generated by oxidation of the olefinic double bond of the sphingosine moiety, whereas for gangliosides the carboxyl group of sialic acid was used to couple with aminopropyl silica gel in the presence of a carbodiimide. These compounds were used for purifying anti-glycolipid antibodies from serum of immunized rabbits. The antibodies bound to the su-strate were released by 2 M potassium thiocyanate and their immunological properties were studied. Aminopropyl silica gel may be preferred over conventional organic solid supports for the following reasons: 1) faster flow rate; 2) higher capacity; 3) easier handling; 4) more economical; and 5) lower susceptibility to microbial attack.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4 degrees C, as well as in silica gel granules at 20 and -60 degrees C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at -60 degrees C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage temperatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at -60 degrees C.     

Novel method for aroma recovery from the bioconversion of lutein to β-ionone by Trichosporon asahii using a mesoporous silicate material

In this work, we report on the synthesis and ability of the mesoporous material MCM-41 to adsorb the norisoprenoid β-ionone. This compound, with a violet aroma note, can be produced from lutein by the yeast Trichosporon asahii through a bioconversion process. We found that β-ionone inhibited the yeast growth and constrained aroma formation. Growth inhibition was overcome using silicate MCM-41 as sorbent device in a fermentation system that allowed product removal from the culture medium by headspace manipulation. Compared to a commercial silica gel, the mesoporous material exhibited a 4.5-fold higher β-ionone adsorption. Contrasting to cultures without the sorbent device, the presence of MCM-41 allowed a marked increase (14-fold) in β-ionone production. Our results suggested that confinement of the norisoprenoid into the sorbent material bypassed its toxicity which allowed a better β-ionone production. This study represents the first report on the use of MCM-41 to recover an aroma produced by fermentation and therefore, a novel application for a mesoporous material.     

Glycolipids of the fish testis.

Glycolipids were purified from the total lipid extract of the testis or milt of a kind of puffer (Fugu rubripes rubripes) by adsorption column chromatography using silicic acid and magnesium silicate and by preparative silica gel TLC. The glycolipids were identified as glucosylceramide (116 mug/g wet tissue) and galactosylceramide 26.7 mug/g). Seminolipid, a sulfagalactolipid specific to mammalian testis was not detected, but the presence of a small amount of sulfatide (15.2 mug/g) was demonstrated. The long-chain bases of both cerebrosides were mainly C18-sphingenine, but in sulfatide, C20-sphingenine was more abundant than C18-sphingenine. In both cerebrosides and sulfatide, the fatty acid compositions were similar, with nervonic acid as the predominant component. Two species of gangliosides were also obtained and were identified as N-acetylgalactosaminyl(1 leads to 4)[N-acetylneuraminyl(2 leads to 3)]galactosyl(1leads to 4)glucosylceramide (59.8 mug/g) and N-acetylneuraminyl(2 leads to 3)galactosyl(1 leads to 4)N-acetylglucosaminyl(1 leads to 3)galactosyl(1 leads to 4)glucosylceramide (45.0 mug/g). The long-chain bases of the two gangliosides consisted of C18-spingenine and C20-sphingenine, and the major fatty acids were palmitic and stearic acids.     

Novel injectable pH and temperature sensitive block copolymer hydrogel

A novel pH and temperature sensitive block copolymer was prepared by adding pH sensitive moiety to temperature sensitive block copolymer. This block copolymer solution showed a reversible sol-gel transition by a small pH change in the range of pH 7.4-8.0 and also by the temperature change in the region of body temperature. The very precise molecular weight control of block copolymer and the prudential tuning of hydrophilic-hydrophobic balance were needed to control the phase diagram. This block copolymer solution forms a gel at 37 degrees C, pH 7.4 (human body). When the block copolymer solution is at room temperature and pH 8.0 as a sol state, both the temperature and pH change are needed for the gelation. This material can be employed as injectable carriers for hydrophobic drugs and proteins, etc. Gelation inside the needle can be prevented by an increase in the temperature during injection, because it does not change into the gel form with only increasing temperature. This material can be used for even a long guide catheter into the body. The block copolymer hydrogel which shows the sol-gel transition by the small pH change from pH 8.0 to pH 7.4 has merits in the delivery system for protein and cells which show cytotoxicity in acidic (below pH 6.5) or basic (above pH 8.5) conditions. This block copolymer system could be used as a template technology for injectable delivery systems.     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.     

Bionanocompositional chitosan-silica sorbent for liquid chromatography

Preparation of hybrid nanocompositional chitosan/silica sorbent was carried out. It was shown that formation of gel in sol-gel process of hydrolytic polycondensation of tetraethoxysilane (TEOS) with including of chitosan consists of two stages. Suppression of crystallization of chitosan in obtained two-phase system and changes in IR spectra are evidenced of interactions between molecules of chitosan and silanol groups of silica network. The resulting hybrid chitosan/silica sorbent was tested by high-performance liquid chromatography (HPLC).     

Reevaluation of the Role of Gangliosides as Receptors for Tetanus Toxin

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Tris-saline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GD1b approximately equal to GT1b approximately equal to GQ1b greater than GD2 greater than GD3 much greater than GD1a approximately equal to GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.     

Effect of silica type and grafting method on the reactivity of tetraneopentylchromium(IV) towards and on silica

The grafting of tetraneopentylchromium(IV) onto the surface hydroxyls of silica has been investigated by varying both the method of deposition and the nature of the silica support. Reaction of the volatile organometallic with the silica surface in vacuo is compared with reaction in solution. A faster reaction but a lower ultimate Cr loading was obtained using the solution deposition technique. The reactions of the organometallic compound with Aerosil 200, a fumed silica often used to model catalyst supports, and Sylopol 952, a silica gel used as a carrier for chromium-based ethylene polymerization catalysts, are compared. After thermal pretreatment at 200 °C, grafting on both supports yields bis(neopentyl)chromium(IV) fragments regardless of the grafting method used, suggesting that a paired arrangement of the surface hydroxyl groups exists on both types of silicas. The higher Cr loading achieved on the silica gel is attributed to its higher surface area. Thermally-induced neopentane elimination from the grafted bis(neopentyl)chromium(IV) fragments occurs at the same rate and with the same stoichiometry for both Aerosil- and Sylopol-supported materials. Consequently, interactions of the grafted organometallic fragments with nearby siloxanes appear to be unimportant in the early stages of the transformation of bis(alkyl)chromium(IV) to the alkylidene.     

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.     

Comparison of air-drying methods for evaluating the desiccation tolerance of liquid culture-produced blastospores of Paecilomyces fumosoroseus

Various drying methods were tested to identify a standard procedure for evaluating the desiccation tolerance of liquid culture-produced blastospores of Paecilomyces fumosoroseus. Since our work is focused on optimizing fermentation conditions for the production of P. fumosoroseus, the criteria for selecting a drying method included ease of use, moderate spore survival after drying and limited variation in spore survival. Three air-drying methods were tested: P. fumosoroseus blastospores mixed with silica gel, with sand, or with diatomaceous earth. Humidity controlled drying was used in the diatomaceous earth drying method. Blastospore survivals after drying were 19% (C.V. range, 32 to 45%), 82% (C.V. range, 26 to 43%), and 2% (C.V. range 32 to 50%) for the silica gel, sand, and diatomaceous earth methods, respectively. Blastospores dried using the silica gel and sand methods had been rinsed in 0.7 m polyethylene glycol before drying and rehydrated in the same solution for determination of survival. The variation observed within each method was similar. The silica gel drying method was selected as most appropriate for our studies based on moderate blastospore survival (19%) and ease of use.     

Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors

The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography-mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4°C, as well as in silica gel granules at 20 and ?60°C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at ?60°C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage tempeatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at ?60°C.     

猪脑中提取高纯度神经节苷脂

应用凝胶层析和离心液相层析法,从猪脑中提取高纯度神经节苷脂(Gls).按脂结合唾液酸(LBSA)计为30.1%,经硅胶薄层层析,580nm扫描结果表明含5种Gls,即:GM1为19.5%,GD3为13.8%,GD1a为27.8%,GD1b为14.2%和GT1b为19.3%.     

Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard

A highly sensitive method for quantification of sialic acids in gangliosides was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedioxy)benzene (DMB) and separated on a reversed-phase C18 column with an isocratic elution. As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived from the reagents. When gangliosides were subjected to acid hydrolysis, the velocity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore gangliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was synthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation. Folch partition was used to decrease lipophilic materials included in the sample, and the sialic acids released were recovered from the upper phase. The present method has a satisfactory sensitivity in the simultaneous quantification of NeuAc and NeuGc in purified gangliosides as well as in crude lipid fractions containing a variety of gangliosides.     

Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment

A method for the detection of GM1b-type gangliosides in complex mixtures of gangliosides was developed. The procedure involves separation of gangliosides on high-performance thin-layer chromatography plates, fixation of the silica gel, treatment with neuraminidase from Vibrio cholerae in the absence of detergent, and incubation of the plates with GgOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies are used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolylphosphate. The procedure is capable of detecting as little as 30 ng of gangliosides. Gangliosides from murine T lymphocytes and from human brain served as examples. Besides GM1b, GD1 alpha is also detectable by this method, whereas the human brain gangliosides GM1a, GD1a, GD1b, and GT1b are not, because they are neuraminidase resistant. Since terminally sialylated gangliosides such as GM1b were described as virus receptors, and certain other terminally sialylated gangliosides are discussed as tumor markers, this method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small amounts of material are available.