Analysis of Volatile Components in Rice Bran

Volatiles stripped from rice bran are analyzed by gas chromatography and thin-layer chromatography either directly or after converted into 2, 4-DNPH’s or 3, 5-DNB’s. Most of prominent peaks revealed by a direct gas chromatography of the volatiles condensates behave as alcohols against classification reagents and are identified as methanol, ethanol, n-propanol, sec-butanol, isobutanol, n-butanol, isopentanol, n-pentanol and n-hexanol, respectively. Ethanol and methanol, being major components in the condensates are also detected by thin-layer chromatography of their 3, 5-DNB’s. Carbonyls identified by a flash exchange gas chromatography of 2, 4-DNPH’s of the volatiles are ethanal, propanal, isobutanal, acetone, n-butanal, isopentanal, n-pentanal and n-hexanal. Thin-layer chromatography of 2, 4-DNPH’s reveals presence of ethanal, acetone, isopentanal or/and isobutanal and n-hexanal in the volatiles. The experimental data have been integrated to furnish a basis for extensive comparison with other rice products and by-products.     

Metabolism of O-Ethyl S,S-Diphenyl Phosphorodithioate (Hinosan®) by Mycelial Cells of Pyricularia oryzae

Metabolism of organophosphorus fungicide Hinosan^® (O-ethyl S, S-diphenyl phosphorodithioate) by mycelia of P. oryzae, rice blast fungus, was studied using ³²P–, ³⁵S– and non-labeled compounds, by ion exchange chromatography, paper chromatography, thin-layer chromatography and gas chromatography, and identifying the metabolites and their derivatives with authentic compounds.

The main metabolic pathway is hydrolysis of one P-S linkage followed by the other P-S linkage or ethyl ester linkage and finally yielding phosphoric acid. A part of the fungicide metabolizes to hydroxylated intermediate metabolite, O-ethyl S-p-hydroxyphenyl S-phenyl phosphorodithioate. No significant difference in rate and mode of metabolism was found in this experiment between susceptible and resistant clones against the fungicide.     

Separation of meso and racemic 2,3-butanediol by aqueous liquid chromatography

Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724) producers meso and nonmeso 2,3-butaneodiol. The enzyme Kinetic of 2,3-butanediol stereoisomer formation from acetone is currently under study in our laboratory. Modeling of these kinetics requires resolution of meso and racemic 2,3-butanediol and positive identification of these resolved components. We report their resolution by aqueous liquid chromatography on both an analytical and a preparative scale. The resolved stereoisomer were identified by a combination of gas chromatography, gas chromatography/mass spectroscopy, ¹³C-NMR spectroscopy, optical activity, and, melting points of the m-dinitrobenzoyl eaters of meso and racemic 2,3-butanediol. An aqueous liquid chromatographic technique for resolving and qualifying major components of a butanediol fermentation mixture in 40 min is presented.     

Fruiting of Schizophyllum commune Induced by Certain Ceramides and Cerebrosides from Penicillium funiculosum

Fruiting bodies were formed around a Penicillium colony which appeared as a contaminant in a culture of Schizophyllum commune, and this phenomenon was reproduced with a synthesized system consisting of S. commune IAM 9006 and P. funiculosum A-l. The active substances were recovered in an acetone extract of the mycelia of P. funiculosum, purified by silica gel column chromatography and reverse-phase high-performance liquid chromatography, and characterized by infrared spectroscopy, gas-liquid chromatography, gas-liquid chromatography-mass spectroscopy and nuclear magnetic resonance spectroscopy. They were ceramides and cerebrosides having nonadecasphingadienine and 2-hydroxy fatty acid moieties in common. The major component was identified as (4E,8E)-N-2^′-hydroxy-(E)-3^′-octadecenoyl-1-O-β-glucopyranosyl-9-methyl-4,8-sphingadienine.     

Synthesis of Sialyllactose from N-Acetylneuraminic Acid and Lactose by a Neuraminidase from Arthrobacter ureafaciens

Two sialyllactose isomers, NeuAcα2→6Galβ1→4Glc and Galβ1→4(NeuAcα2→6)Glc, were prepared by incubation of a concentrated solution of N-acetylneuraminic acid and lactose in the presence of a neuraminidase from Arthrobacter ureafaciens. Each sialyllactose was isolated by a combination of ion-exchange chromatography and high performance liquid chromatography. The structure of each sialyllactose was identified by mass spectrometry, nuclear magnetic resonance spectrometry, and enzymatic analysis.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

Overexpression and Purification of Rhizobium etli Glutaminase A by Recombinant and Conventional Procedures: A Comparative Study of Enzymatic Properties

Rhizobium etli glutaminase A was purified to homogeneity by conventional procedures that included ammonium sulfate differential precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, and dye-ligand chromatography. Alternatively, the structural glsA gene that codifies for glutaminase A was amplified by PCR and cloned in the expression vector pTrcHis. The recombinant protein was purified to homogeneity by affinity chromatography. This protein showed the same kinetic properties as native glutaminase A (K_m for glutamine of 1.5 mM and V_max of 80 μmol ammonium min⁻¹ mg protein⁻¹). Physicochemical and biochemical properties of native and recombinant glutaminase were identical. The molecular mass of recombinant glutaminase A (M_r 106.8 kDa) and the molecular mass of the subunits (M_r 26.9 kDa) were estimated by mass spectrometry. These results suggest that R. etli glutaminase A is composed of four identical subunits. The high-level production of recombinant glutaminase A elevates the possibilities for determination of its three-dimensional structure through X-ray crystallography.     

Determination of 4-chloroindole-3-acetic acid methyl ester in Lathyrus, Vicia and Pisum by gas chromatography – mass spectrometry

4-Chloroindole-3-acetic acid methyl ester was identified unequivocally in Lathyrus latifolius L., Vicia faba L. and Pisum sativum L. by thin layer chromatography, gas chromatography and mass spectrometry. The gas chromatographic system was able to separate underivatized chloroindole-3-acetic acid methyl ester isomers. The quantitative determination of 4-chloroindole-3-acetic acid methyl ester in immature seeds of these three species was performed by gas chromatography – mass spectrometry using deuterium labelled 4-chloro-indole-3-acetic acid methyl ester as an internal standard. P. sativum contained approximately 25 mg kg^-1, V. faba 1–2 mg kg^-1 and L. latifolius 2 mg kg^-1 dry weight.     

The Systematic Separation and Identification of Pesticides in the First Division

The systematic identification and determination of many kinds of pesticides were tried by a combination of column, thin-layer and gas chromatography. Pesticides in the first division,¹⁾ e.g., aldrin, DMC ethylene, CPAS, DDDS, o,p′-DDT, p,p′-DDT, heptachlor, quintozene, Telodrin and tetrasul, were separated from pesticides in the second division,¹⁾ e.g., α-BHC, β-BHC, γ-BHC, trifluralin, CPA, CNP, nitrofen, endrin, dieldrin, dicofol, etc. by column chromatography. Further, pesticides in the first division were separated from each other and determined approximately by thin-layer and gas chromatography.

The recoveries of these pesticides, except for aldrin and CPAS, were about 100%. The recoveries of aldrin and CPAS were about 70 %, due to decomposition during the procedures, or to adsorption on the adsorbent during the column chromatography. It was presumed from GC-MS data that CPAS was decomposed to DDDS, tetrasul and other compounds during the procedures.     

DETECTION OF CHLOROPHYLLS c1, c2 AND c3 IN PIGMENT EXTRACTS OF PRYMNESIUM PARWM (PRYMNESIOPHYCEAE)1

Three chlorophyll c-type pigments were separated by reversed-phase high Performance liquid chromatography and thin-layer chromatography from pigment extracts of the prymnesiophyte, Prymnesium parvum Carter. Based on spectral characteristics, retention times, and comparison with reference pigments isolated from the diatom Phaeodactylum tricornutum Bohlin, two of these pigments were identijied as chlorophyll c₁ and c₂. The other pigment was identified by its absorption spectrum and thin-layer chromatography retention times as the newly described chlorophyll c₃. However, in other prymnesiophytes so far examined, chlorophyll c₁ and chlorophyll c₃ were present with no chlorophyll c_l. The discovery of chlorophyll c₃ with chlorophyll c₁ and chlorophyll c₃ in Prymnesium parvum therefore represents the first report of this combination of pigments in prymnesiophytes.     

Biotransformation and toxicity of aniline and aniline derivatives in cyanobacteria

Agmenellum quadruplicatum strain PR-6 and Oscillatoria sp. strain JCM grown photoautotrophically in the presence of aniline metabolized the aromatic amine to formanilide, acetanilide and p-aminophenol. The metabolites were isolated by either thin-layer, gas-liquid or high pressure liquid chromatography and identified by comparison of their chromatographic, ultraviolet absorbance and mass spectral properties with those of authentic compounds. The toxicity of aniline derivatives towards Agmenellum quadruplicatum strain PR-6 indicated that the cyanobacterium was extremely sensitive to o-, m- and p-aminophenols, and phenylhydroxylamine.Abbreviations TLC thin layer chromatography - HPLC high pressure liquid chromatography - GC/MS gas chromatography/mass spectrometry - m/e mass to charge ratio     

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver -glucuronidase was studied by sequential lectin affinity chromatography. -Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed byN-[¹⁴C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography.Ulex europaeus agglutinin-agarose chromatography revealed the presence of (1-3) linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin,Ricinus communis agglutinin andPhaseolus vulgaris erythroagglutinin.     

Isolation and preliminary characterisation of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix)

Two toxic phospholipases A have been isolated from the venom of the Malayan cobra (Naja naja sputatrix). The phospholipases A were purified by successive ion-change chromatography on SP-Sephadex C-25, Sephadex G-75 gel filtration chromatography and successive Bio-Rex 70 ion-exchange chromatography. The purified toxic phospholipases A were homogeneous electrophoretically. They were designated as sputatrix phospholipase A-I and sputatrix phospholipase A-II. Positional specificity studies showed that they belong to the A₂-type phospholipase A. The medium lethal dose 50% (LD₅₀) values of the two phospholipases A are 0.27 and 0.28 μg/g, respectively, by intravenous injection and 1.05 and 1.00 μg./g, respectively, by intraperitoneal injection. The molecular weights of the two enzymes are 14 000 as determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Amino acid composition of sputatrix phospholipase A-I differs from sputatrix phospholipase A-II only by having one extra amino acid: a glutamic acid. Amino acid compositions of the two enzymes are also similar to those of other cobra venom phospholipases A.     

Amylostatins,Other Amylase Inhibitors Produced by Streptomyces diastaticus subsp. Amylostaticus No. 2476

Amylase inhibitors (amylostatins) other than those reported as S-AI were found in the culture filtrate of Streptomyces diastaticus subsp. amylostaticus No. 2476. They were separated grossly into F-1a, F-1b and F-2 fractions by column chromatography on Dowex 50W × 4 (NH₄⁺) and by preparative high performance liquid chromatography. Each fraction was further separated by preparative paper partition chromatography (PPC). Fractions obtained by PPC had different inhibitory activities against various amylases. On the other hand, acid hydrolysis of each active inhibitory fraction produced amylostatin X′ (C₁₃H₂₁NO₇) and/or amylostatin XG (C₁₉H₃₃NO₁₃). The diversities and common features of these amylostatins are discussed.     

Isolation of the trypsin inhibitors in Ascaris lumbricoides var. suum using affinity chromatography

A method for isolating three water-soluble trypsin inhibitors from Ascaris lumbricoides var. suum by affinity chromatography is described. The trypsin inhibitors captured by affinity chromatography are resolved into three species by chromatography on CM-Sephadex at pH 8.1. The inhibitors are named in the order that they are released from the CM-Sephadex column. Ascaris Trypsin Inhibitor 1 is the same as inhibitor CM-1 described by [3.] and inhibitor Peak I of U. Kucich and [4.]. Ascaris Trypsin Inhibitor 2 is the inhibitor described by [2.] and inhibitor CM-2 of [3.]. Ascaris Trypsin Inhibitor 3 is the same as inhibitor Peak II of [4.]. Ascaris Trypsin Inhibitor 1 is 80%, Ascaris Trypsin Inhibitor 2 is 8%, and Ascaris Trypsin Inhibitor 3 is 12% of the water-soluble trypsin inhibitors present in Ascaris. With this procedure all of the Ascaris trypsin inhibitors can be isolated in a few days. This shortens the exposure of personnel to crude extracts of Ascaris and diminishes the biological hazard of working with them. Frequent exposure to Ascaris extracts may evoke an anaphylactic response in personnel.     

高良姜地上部分黄酮类成分的研究

为了解高良姜(Alpinia officinarum Hance)中的化学成分,采用硅胶柱色谱和葡聚糖凝胶柱色谱从高良姜地上部分分离得到5个黄酮类化合物。经光谱分析分别鉴定为:高良姜素(1)、高良姜素-3-甲醚(2)、乔松素(3)、二氢高良姜醇(4)和山奈酚(5)。     

Enzymatic Formation of d-Pantothemc Acid-β-Glucoside by Various β-Glucosidases

A β-gIucoside of d-pantothenic acid was formed from d-pantothenic acid and β-glucosyl donors such as cellobiose, phenyl-β-d-glucoside, salicin, and 4-methylumbelliferyl-β-d-glucoside and naphthol AS-BI-β-d-glucoside by various β-glucosidases, i.e., almond β-glucosidase, cellulase type II and III, naringinase, and hesperiginase. The compound was isolated from a reaction mixture of almond β-glucosidase by treatment with active charcoal, Amberlite CG–50, and DEAH-cellulose column chromatography, paper chromatography, and Sephadex G-IO gel filtration. Then, the compound was characterized as 4′-O-(β-d-glucopyranosyl)-d-pantothenic acid by various analytical methods including bioassay, paper chromatography, NMR and specific optical rotation. The microbiological activities of the compound were also determined.     

Studies on γ Globulin of Rice Embryo

All globulin components hitherto found in many species of seeds, α, β, γ and δ globulins, were identified in rice grain by ultracentrifugal experiments and gel-filtration chromatography. Among them, γ globulin was found to occur in high concentration in embryo and bran which were the most active parts in biological functions of rice grain. Then γ globulin was isolated from embryo by gel-filtration chromatography on a Sephadex G-200 column. Purified γ globulin was homogeneous in ultracentrifugal analysis and it was found to be insoluble in cold saline solution. On the other hand, α and β globulins were found to be more concentrated in endosperm with considerable heterogeneity.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

A plant lipid and the platelet-activating factor stimulate ATP-dependent H+ transport in isolated plant membrane vesicles

A plant lipid was isolated from zucchini (Cucurbita pepo L.) membranes and from soybean (Glycine max [L.] Merr) phospholipids by thinlayer chromatography and further purified by high-performance liquid chromatography. This plant lipid was chromatographically very similar to the platelet-activating factor, an ether phospho-lipid with hormone-like properties found in mammals. Both the plant lipid and the platelet-activating factor stimulated ATP-dependent H⁺ transport in isolated membrane vesicles from zucchini hypocotyls.Abbreviations HPLC high-performance liquid chromatography - PAF platelet-activating factor