Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune

Isolation and identification of substances having an activity to stimulate the fruiting body formation of Schizophyllum commune were attempted. The active principles in its mycelia were divided into four fractions by sequential purification with silica gel column and reverse-phase HPLC column chromatography. By infrared spectra and thin-layer chromatography, the active substances in these four fractions were revealed as cerebrosides. About 0.1 μg of the cerebroside fractions showed a discriminative stimulating activity on S. commune when tested by the method these authors adopted. The active substance in the fraction II was N-2′-hydroxypalmitoyl-1-O-glucosyl-nonadecasphingadienine. The cerebrosides from pea seeds and Fusicoccum amygdali showed the similar activity on S. commune, but some commercial synthetic cerebrosides and cerebrosides from bovine and porcine brains exhibited no stimulating activity. Only definite cerebrosides with special structures seem to be able to induce the fruiting of S. commune.     

A method for fractionation of cerebrosides into classes with different fatty acid compositions

A method is described for the separation of beef brain cerebrosides into three fractions containing different classes of fatty acids: nonhydroxy (I), unsaturated nonhydroxy (II), and hydroxy fatty acid cerebrosides (III). The procedure consists of benzoylation of either crude or purified cerebrosides, followed by column chromatographic separation of benzoylated derivatives containing nonhydroxy acids from those containing hydroxy fatty acids. The benzoyl groups are removed by sodium methoxide-catalyzed transesterification; from the reaction mixtures, fractions I and III precipitate. The fraction II present in mother liquor of I was shown to contain mainly short-chain and unsaturated nonhydroxy fatty acid cerebrosides. The fatty acid composition of each fraction was obtained by gas-liquid chromatography.     

Cerebrosides of human aorta: isolation, identification of the hexose, and fatty acid distribution

Cerebrosides have been isolated from adult human aortic tissue. Each aorta was divided into portions classified as normal, fatty streaks, fibrous plaques, or complicated lesions. The cerebrosides were isolated by Florisil column chromatography, mild alkaline methanolysis, a second Florisil column, and preparative thin-layer chromatography. The concentration of cerebrosides was higher in fatty streaks than in the more advanced plaques; apparently normal tissue gave the same cerebroside content as plaques found in the same aorta. The quantities of cerebrosides ranged from 0.01 to 0.73% of the total lipid. Of the 16 cerebroside samples isolated, 10 contained glucosyl ceramide, 1 contained galactosyl ceramide, and 5 were not analyzed for specific hexose. The fatty acid distribution was determined for 11 of the samples; it was similar to that of spleen cerebrosides. We suggest that aortic cerebrosides originate in the plasma. "Normal tissue" cerebrosides contained less unsaturated fatty acid than cerebrosides from a diseased area of the same aorta. Preparative thin-layer chromatography, the last step of cerebroside isolation, always separated at least two unidentified substances. One of these substances yielded both glucose and galactose on acid hydrolysis. Their removal from the cerebrosides accounts for the lower values for cerebroside compared to other authors' determinations.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

A Large Scale Procedure for Purification of Desmosine and Isodesmosine

A rapid large scale procedure was devised for the purification of desmosine and isodesmosine from ligamentum nuchae elastin. The method makes use of the hydrophilic nature of the desmosines which preferentially absorbs to cellulose fibers in mixtures of organic solvents. Resolution of the isomers was achieved on a polystyrene resin column.     

A large scale procedure for purification of desmosine and isodesmosine.

A rapid large scale procedure was devised for the purification of desmosine and isodesmosine from ligamentum nuchae elastin. The method makes use of the hydrophilic nature of the desmosines which preferentially absorbs to cellulose fibers in mixtures of organic solvents. Resolution of the isomers was achieved on a polystyrene resin column.     

Cerebrosides of<Emphasis Type="Italic"> Candida lipolytica</Emphasis> yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms. The dominant fatty acid was h16:0. The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine. The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides. The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.     

Simultaneous determination of psychosine and cerebrosides

A new method was developed for the simultaneous determination of psychosine and cerebrosides in tissues. Total lipids extracted from the tissues were treated with [3H]acetic anhydride in toluene-methanol. Known amounts of nonradioactive N-acetylpsychosine were added to the reaction product and then subjected to mild alkaline methanolysis. After the product was washed, it was fractionated by silica gel column chromatography and the fraction containing glycolipids was benzoylated. The benzoylated product was finally fractionated on TLC. The amounts of benzoylated derivatives of nonhydroxy- and hydroxycerebrosides and N-acetylpsychosine were determined using a scanning densitometer. The amounts of psychosine in tissues were calculated from the radioactivity in the spot of N-acetylpsychosine and the recovery of added carrier N-acetylpsychosine. This method allowed us to determine 5 to 1000 pmol of psychosine and 1 to 20 nmol of cerebrosides in peripheral nerves and other tissues of the twitcher mouse as well as transfected Schwann cells derived from the sciatic nerves.     

Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm.

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.     

Acid beta-galactosidase from human fibroblasts. A microscale purification method monitored by a highly sensitive enzyme assay

A highly sensitive microassay method and a microscale purification system were developed to isolate the residual acid beta-galactosidase in GM1-gangliosidosis fibroblasts. The sensitivity of the microassay system, composed of a 96-well microplate and a microplate fluorometer, was 100-fold higher than that of the conventional system and the response was linear in the pmole range. Acid beta-galactosidase was characterized as a thiol enzyme which was inactivated by a mercuric compound. This enzyme was completely adsorbed on an Hg-agarose column and was easily eluted from the column by 10 mM 2-mercaptoethanol. The microscale purification system using Con A-Sepharose, PAT-Sepharose, and Hg-agarose column chromatography achieved 565- and 7,970-fold purifications of acid beta-galactosidase with an overall yields of 44% and 45% from normal and GM1-gangliosidosis fibroblasts, respectively. The purified enzyme fractions did not contain any other lysosomal enzyme activities except for a small amount of beta-N-acetylhexosaminidase activity.     

Pre-packed reverse phase columns for isolation of complex lipids synthesized from radioactive precursors

Pre-packed reverse phase columns (Bond Elut) were used for the separation of complex lipids, such as phosphatidylcholine, cerebrosides, sulfatides, and gangliosides, from their respective water-soluble radioactive precursors after their in vitro biosynthesis. After an incubation in vitro, the entire reaction mixture is passed through the column, where complex lipids are retained and the hydrophilic radioactive precursors are washed away from the column. The retained lipids are then eluted with a more nonpolar organic solvent. The procedure is shown to be simpler and more efficient than the normally used Folch partitioning method or other procedures.     

Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.     

The extractive purification of peroxidase from plant raw materials in aqueous two-phase systems

The extractive purification of peroxidase from Armoracia rusticana roots and Glycine max seed coats in temperature-induced and affinity microsphere-containing aqueous two-phase systems was stuied. The extractive purification of peroxidase from Glycine max seed coats was carried out in a temperature-induced aqueous two-phase system formed by Triton X-45, Triton X-100 and sodium acetate at pH 5.5 A 99% yield with a 6-fold purification factor was obtained. When the clear top phase was subjected to concanavalin-A affinity chromatography, the purification factor rose to 41 and the yield dropped to 28%. A two-step purification process for peroxidase from Armoracia rusticana roots was developed by adding concanavalin-A affinity microspheres to a PEG/phosphate aqueous two-phase system. The method allows a 60% recovery of high purity peroxidase (1,860 guaiacol units per mg). A lower recovery rate and degree of purification of this enzyme was achieved after temperature-induced aqueous two-phase partition or acetone precipitation and concanavalin-A affinity column chromatography.     

Separation of phytanic and pristanic acid by high-pressure liquid chromatography: application of the method

The synthesis of pristanic acid from phytanic acid, and a simple reversed-phase high-pressure liquid chromatographic (HPLC) method for the separation and purification of these acids, is described. A base-line separation of [U-3H]phytanic and [U-3H]pristanic acid is achieved with a graphitized carbon column. The isoprenoid metabolites formed after incubation of cultured fibroblasts with phytanic or pristanic acids are extracted with a Sep-Pak C18 cartridge and separated from the substrates by the same reversed-phase HPLC used for substrate purification. The methods are suitable for studies on the mechanisms for degradation of phytanic acid. Recently, different inborn errors with accumulation of phytanic acid have been defined. The present method will be a useful tool in our efforts to define these metabolic defects and their subcellular localization.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

Determination of aldosterone secretion rate utilizing mixed mode and high performance liquid chromatography

A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates.     

The Azure Dyes their Purification and Physicochemical properties. II. Purification of Azure B

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or plychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polyineric adsorbent (XAD-2) the acetate-formate anions are exchanged for chloride. Regeneration of both columns is possible: KMnO₄, Na₂S₂O₄ and water are run through column A, 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

Preparative high speed gel permeation chromatography of proteins on Toyopearl HW55F

A method is described for the rapid separation of protein mixtures by high speed gel permeation chromatography using Toyopearl HW55F, a new semi-rigid hydrophilic polymer. Good resolution of protein mixtures according to molecular size can be achieved on this material with high flow rates and low column pressures. Molecular weight estimations in the range between 10⁴ and 10⁶ daltons can be performed within minutes. Large-scale enzyme purification (up to 1.6 gm of starting material with a 3.2 × 105 cm column) was achieved with 86–110% recovery of enzymatic activity. Data are presented on the optimum column length, flow rate, loading capacity and eluant ionic strength.     

Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles

Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis. We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution. Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles. The reconstituted receptors still bound radioligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors.