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Strain SM1 was isolated as a biosurfactant-producing microorganism from seawater and presumptively identified as Myroides sp., based on morphology, biochemical characteristics and 16S rDNA sequence. The strain produced surface-active compounds in marine broth, which were purified, using emulsification activity for n-hexadecane as an indicator. The purified compounds were identified by thin-layer chromatography, (1)H- and (13)C-NMR spectra and fast atom bombardment mass spectrometry as cholic acid, deoxycholic acid and their glycine conjugates. Type strains of the genus Myroides, M. odoratus JCM7458 and M. odoramitimus JCM7460, also produced these compounds. Myroides sp. strain SM1 possessed a biosynthetic route to cholic acid from cholesterol. Thus, bile acids were found as new products of prokaryotic cells, genus Myroides.

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OBJECTIVE: To find out the optimal extraction technique for HDCA (a-hydroxycholic acid). METHOD: According to the orthogonal design L9(3(4)), the optimal extraction technique was sought through experimental investigation, and the content of HDCA was determined by TLC. RESULT: The optimal extraction method was eightfold 8% NaOH solution and 16 hours. The optimal purification method was six fold ethyl acetate, 5% active carbon, and 30 minutes twice. CONCLUSION: The above mentioned extraction technique is optimal and feasible in extraction of HDCA.

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OBJECTIVE: To investigate the mechanism of Qingkailing (QKL) Injection in treating acute promyelocytic leukemia. METHODS: Using MTT technique, cell morphologic method, DNA gel electrophoresis and flow-cytometry to study the human acute promyelocytic leukemia (HL-60) cell apoptosis induced by QKL and its active principle. RESULTS: QKL and its active principle, Baicalin and hyodeoxycholic acid, showed strong cytotoxicity in inhibiting HL-60 cell, the Bezoar cholic acid showed a weaker effect. Apoptosis could be induced after being treated for 6 hrs by the former three principles, showing a typical apoptosis peak under flow-cytometry, but could not be induced by the latter one. CONCLUSION: QKL could induce leukemia cell apoptosis in vitro, which may be one of the mechanisms of QKLI in curing acute promyelocytic leukemia.

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A simple and efficient method for the separation of individual unconjugated bile acids and their glycine- and taurine-amidated, 3-sulfated, 3-glucosylated and 3-glucuronidated conjugates is described. The method involves the use of a two-dimensional (2D) reversed-phase (RP) high-performance thin-layer chromatographic (HPTLC) technique with methyl beta-cyclodextrin (Me-beta-CD). Five major unconjugated bile acids, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid and lithocholic acid, and their conjugates were examined as the solutes. A high degree of separation of individual bile acids in each homologous series was achieved on a RP-HPTLC plate by developing with aqueous methanol in the first dimension and the same solvent system containing Me-beta-CD in the second dimension. In particular, all of the six 'difficult-to-separate' pairs, unconjugated CDCA and DCA and their conjugated forms with glycine, taurine, sulfuric acid, D-glucose and D-glucuronic acid, were effectively resolved by adding Me-beta-CD in the aqueous mobile phases with the formers having larger mobilities than the latter. The application of this 2D inclusion RP-HPLC method to the separation of glycine-conjugated bile acids in human bile is also described. The present method would be useful for separating and characterizing these bile acids present in biological materials.

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The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.

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A simple and rapid technique is described for the removal of Triton X-100, deoxycholate, and cholate from protein solutions. The method involves a 2-min centrifugation of the sample on a Bio-Beads SM-2 bed prepared in a microcentrifuge tube and is suitable for multiple assays of 0.05- to 0.45-ml samples. Another advantage of this method is the high recovery of proteins without dilution of the sample.

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A simplified extraction procedure for bile acids from wet faeces, using methanol/hydrochloric acid is described. Extracts were analyzed by gas-liquid chromatography, thin-layer chromatography and an enzymatic assay, with 3 alpha-hydroxysteroid dehydrogenase. Recoveries of some stable bile acids, added to faeces, were studied; extraction efficiency was also investigated with a procedure using radioactive labelled bile acids given orally to patients. Resin treatment of faecal extracts, because of the sometimes hard colour of the extracts, resulted in a slightly lower recovery as determined by the enzymatic method. Recoveries were higher, using the proposed extraction procedure, than those obtained with extracts prepared by the standard procedure of Grundy et al [6].

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The ability of canine pancreatic signal peptidase to remove the signal peptide portion of presecretory proteins in a translocation-independent assay is shown to require phospholipid. Sodium deoxycholate extracts of canine pancreatic rough microsomes containing both signal peptidase and phospholipid were delipidated by gel filtration chromatography on Sepharose CL-6B equilibrated with 0.2% deoxycholate. Column fractions were assayed for signal peptidase activity both with and without the addition of ethanol-extracted soybean phospholipid at a final concentration of 1.0 mg/ml. A peak of signal peptidase activity was detected only when the fractions were assayed with added phospholipid. Phospholipid assays demonstrated that the peak of signal peptidase activity was cleanly separated from phospholipid. The ratio of protein to phospholipid in the deoxycholate extract of rough microsomes was 1.76 while that of the most active signal peptidase fractions ranged from 46.1 to 138. The peak of signal peptidase activity exhibited an apparent Stokes radius of 55 A. Highly purified preparations of phosphatidylcholine were most effective in restoring activity to delipidated signal peptidase. Phosphatidylinositol was much less effective. Phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine were all ineffective.

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Tauroallocholate is the major bile salt of the lizard, Uromastix hardwickii. Alkaline hydrolysis of bile from 25 gallbladders provided 1.21 g of acidic material, about 90% of which was allocholic acid. Analyses by gas-liquid chromatography, and mass spectrometry verified the presence of almost 10% of deoxycholic acid and smaller amounts of other 5alpha and 5beta-bile acids.

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A column packed with calcium-free bovine aorta elastin provided good separations of mixtures of bile salts when water was the moving phase. Tritium-labelled cholesterol was applied to the column using dilute solutions of taurodeoxycholate in Tris-NaCl buffers as solvent. The cholesterol was quantitatively eluted as a narrow peak in a rising gradient of taurodeoxycholate. When Na+ in the buffer was replaced by Ca2+ elution of the labelled cholesterol was delayed. Control experiments in which the elastin fibres were replaced as the column packing by an inert stationary phase consisting of n-butanol immobilized by silane-treated Celite showed that the effect of the change from Na+ to Ca2+ on the solvent properties of taurodeoxycholate was small and in the opposite direction. The experiments indicated that the replacement of sodium by calcium as the ionic environment of fibrous elastin produced a configurational change towards increasing hydrophobic character.

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