Cell wall composition of Micromonospora olivoasterospora, Micromonospora sagamiensis, and related organisms.

Cell walls of 19 Micromonospora species were analyzed for their components. All the cell walls had xylose and arabinose, but the presence of glucose, galactose, mannose, or rhamnose depended on the strain. Amino acids present in the walls consisted of glycine, glutamic acid, diaminopimelic acid, and alanine, in a molar ratio of approximately 1:1:1:0.6--0.8. 3-Hydroxydiaminopimelic acid, together with meso-diaminopimelic acid, was found in many species and was isolated from Micromonospora olivoasterospora to compare the color constant in an amino acid analyzer with that of meso-diaminopimelic acid. The cell walls of Micromonospora sagamiensis and M. olivoasterospora contained only D-alanine and not L-alanine. All species tested except Micromonospora globosa contained glycolate in an almost equimolar ratio to diaminopimelic acid in their cell walls. Among 45 strains of 12 genera examined, Actinoplanes, Ampullariella, Amorphosporangium, and Dactylosporangium species had a significant amount of glycolate in the whole cells. Based on these results, the primary structure of the peptidoglycan of Micromonospora is discussed.     

Chemical synthesis of 1-O-alkyl and 1-O-acyl dihydroxyacetone-3-phosphate

A method for the chemical synthesis of 1-O-hexadecyl dihydroxyacetone-3-phosphate is described. The synthesis was started with the preparation of O-hexadecyl glycolic acid by condensing 1-iodohexadecane with ethyl glycolate in the presence of silver oxide, followed by saponification and free acid liberation with HC1. O-Hexadecyl glycolic acid was converted to the acid chloride (with oxalyl chloride) which was condensed with diazomethane in diethyl ether to form hexadecyloxy diazoacetone. The diazoketone was decomposed by H₃PO₄ in dioxane to give the desired product, 1-O-hexadecyl dihydroxyacetone-3-phosphate. The product was purified by chromatography on silicic acid column followed by an acid wash. The final yield was 50% starting from O-hexadecyl glycolic acid. Analytical, spectral (IR, NMR) and chromatographic properties of 1-O-hexadecyl dihydroxyacetone-3-phosphate are described. The method described here may be used to prepare different acyl and alkyl derivatives of dihydroxyacetone phosphate in good yield as illustrated by describing the procedure for the synthesis of 1-O-palmitoyl dihydroxyacetone-3-phosphate, 1-O-hexadecyl dihydroxyacetone-3-[³²P] phosphate and the dimethyl ketal of 1-O-palmitoyl [2-¹⁴C]dihydroxyacetone phosphate.     

Expression of active spinach glycolate oxidase in Aspergillus nidulans

The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity. (c) 1996 John Wiley & Sons, Inc.     

Determination of glycolic acid level in higher plants during photorespiration by stable isotope dilution mass spectrometry with double-labeling experiments

Determination of glycolic acid by stable isotope dilution was applied to the measurement of the glycolic acid pool size in tomato and maize leaves during photorespiration. Detached leaves were maintained in the presence of 18O2; [13C]glycolate was added to the foliar extract as an internal standard and the mixture of biological glycolate and [13C]glycolate was analyzed by combined gas chromatography-mass spectrometry. The level of foliar glycolate pool was measured via the 13C label, and 18O incorporation was determined.     

THE EXTRACELLULAR RELEASE Of GLYCOLIC ACID BY A MARINE DIATOM1

The excretion of glycolic acid by the marine diatom Chaetoceros socialis through time was studied. Excretion in axenic cultures was linear for the time intervals used, but for nonaxenic cultures an equilibrium was created, suggesting bacterial uptake of glycolic acid. In studies with an inhibitor of glycolate dehydrogenase, the level of glycolic acid in the medium jumped 15–fold. This shows the presence of this enzyme, and implies the presence of the entire set of enzymes which convert glycolic acid to serine and release carbon dioxide. In both axenic and nonaxenic cultures a steady state was reached. All of the data suggest that at high cell densities glycolic acid is liberated from the cell by a passive mechanism. The effect of such an excretion in natural waters is discussed.     

Identification and quantitation of fatty acid ethyl esters in biological specimens

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.     

Chromatographic analysis of griseofulvin and metabolites in biological fluids

A simple and accurate assay for the determination of griseofulvin and its metabolites in biological fluids using high-performance liquid chromatography is described. Using a reversed phase column and a mobile phase solvent of 45% acetonitrile in 0.1 M acetic acid, baseline separation of griseofulvin and several analogues was obtained. The described method allows one to quantitatively determine griseofulvin, 6-demethylgriseofulvin, and griseofulvic acid, a newly identified metabolite in man, in urine and plasma samples. Treatment of plasma samples prior to the analysis is simply made by deproteinizing the samples with an equal volume of acetonitrile. For urine samples, the procedure involves diethyl ether extraction with subsequent evaporation to dryness and reconstitution with the mobile phase solvent.     

Glycolate Metabolism in Eurasian Watermilfoil (Myriophyllum spicatum)

The metabolism of glycolate by Eurasian watermilfoil (Myriophyllum spicatum L.), a submersed angiosperm, was studied by feeding radioactive glycolate and glyoxylate and by analysis of glycolate and glycolic acid oxidase. Evidence for operation of the glycolate pathway is given. Glycolic acid oxidase occurs at levels comparable to amounts in species showing photorespiration. This species has a high affinity for CO₂ and a possible mechanism for it is described.     

A Method for Fractional Determination of Soybean Sterols in Four Classes by Florisil Column Chromatography

A procedure for fractional determination of soybean sterols is presented. Sterols in lipid extracts were fractionated into four classes, fatty acid esters, the free form, acylated glucosides and non-acylated glucosides, by Florisil column chromatography. Sterol contents in the four classes were determined colorimetrically with ferric chloride-perchloric acid reagent. Before the colorimetry, the fatty acid ester fraction was hydrolyzed with ethanolic KOH, and the sterol was isolated as tomatinide. The free sterol fraction was directly treated with tomatine solution. The tomatinides were dissociated with dimethyl sulfoxide. To avoid the contamination of pigments from the acylated glucoside fraction, the second Florisil column was rinsed with diethyl ether between the elution with the first solvent (0 to 50% diethyl ether in n-heхane) and that with the second solvent (0 to 30% methanol in diethyl ether).     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

Studies on the Residual Respiration not Inhibited by KCN Plus Hydroxamic Acid in Tobacco Callus Cultures

A residual respiration not inhibited by KCN plus hydroxamic acid had been observed in many plant organs and tissues. The relative O2 uptake of it was 20–30% of total respiration in tobacco callus cultures. However, there is no report concerning the nature of the residual respiration and its localization in cell. The object of this study is to elucidate the characteris- tics of this residual respiration and its localization in cell. The experimental results are as follows: 1. The additions of glycolate and glyoxylate cause a marked rise in residual respiration not inhibited by KCN (or NaN3) plus m-CLAM. 2. The O2 uptake induced by glycolate and the residual respiration is inhibited by the addition of α-hydroxy ethanesulfonate. 3. The mitochondrial respiration is completely inhibited by KCN plus m-CLAM, but no effect by adding of glycolate. 4. Oxidation reactions of glycolate and glyoxylate in supernatant are observed after mitochondria are removed. Based on the above results, it is suggested that the residual respiration not inhibited by KCN plus m-CLAM in tobacco callus cultures is primarily catalyzed by glycolic acid oxidase localized within microbodies.     

A radioimmuno—chromatographic scanning method for the analysis of testosterone conjugates in urine and serum

A method for the analysis of testosterone (and 5α-dihydrotestosterone) conjugates in human serum and urine samples is described. The samples were brought to pH 1 and extracted with a diethyl ether—methanol mixture. After evaporation the residues were run in a thin-layer chromatography system, individual samples' paths were cut into 1-cm long pieces and eluted with methanol. The methanol was evaporated and the residue subjected to acid hydrolysis. The released steroid was extracted by diethyl ether and measured by radioimmunoassay. The methodology described represents a new approach to the qualitative and quantitative study of steroid conjugates in serum and urine, and can easily be applied to the study of steroid conjugates in other biological material.     

The specific radioactivity of glycolic acid in relation to the specific activity of carbon dioxide evolved in light in photosynthesizing sunflower leaves

Attached leaves of sunflower (Helianthus annuus L.) were exposed to ¹⁴CO₂ during steady-state photosynthesis for 2 to 30 min in 345 l/l CO₂ and 21% O₂ at 29° C and a light intensity of 1300 E m^-2s^-1. Glycolic acid was extracted with water and diethyl ether, and was determined in the aqueous residue by high-pressure liquid column chromatography. The relative specific radioactivity of the glycolic acid synthesized during photosynthesis reached about 100% after 30 min of photosynthesis and was almost equal to that of the CO₂ evolved during photorespiration, their ratio at all times being nearly one. These results provide strong in-vivo evidence that the glycolic acid is the substrate for CO₂ evolved by sunflower leaves in light.     

Comparative Study of the Cell Walls of the Yeastlike and Mycelial Phases of Histoplasma capsulatum

Cell walls were prepared from the yeastlike and mycelial phases (YP and MP) of Histoplasma capsulatum and from Saccharomyces cerevisiae by mechanical disruption and washing. Lipids were extracted with methanol-ether, chloroform, and acidified methanol:ether; a final extraction was made with ethylenediamine. The lipid contents of H. capsulatum YP and MP walls were about the same. Qualitative and quantitative analyses were made of the products obtained from treatment of the cell walls, or fractions from them, with weak acid or with enzymatic preparations containing glucanase and chitinase activities. YP walls contained much larger quantities of chitin and smaller quantities of mannose and amino acids than the MP walls. H. capsulatum MP was shown to resemble S. cerevisiae by low chitin content and by the presence of a mannose polymer, soluble in ethylenediamine and water. H. capsulatum MP chitin appeared to be intimately associated with glucose in the wall, since enzymatic hydrolysis of the residue after mild acid hydrolysis of cell walls or fractions from them resulted in the release of glucose and acetylglucosamine; only acetylglucosamine was released from YP walls with such treatment. By electron microscopic observations, the unextracted MP cell walls were much thinner than the YP, and neither wall appeared laminated.     

Effect of banana stem juice on biochemical changes in liver of normal and hyperoxaluric rats.

Influence of stem extract of banana (family Musaceae), was studied on glycolic acid oxidase (GAO) and lactate dehydrogenase enzymes, calcium, phosphorus, oxalate and glycolic acid in liver tissues of sodium glycolate-induced hyperoxaluric rats. Activity of GAO was significantly lowered in the extract-treated rats compared to that of the glycolate-fed rats. LDH increased significantly in glycolate administered rats when compared with the extract-treated rats. The levels of calcium, phosphorus, oxalate and glycolic acid during hyperoxaluric state showed remarkable alterations in liver tissue.     

pH Dependence and Kinetics of Glycolate Uptake by Intact Pea Chloroplasts

Experiments in which [1-¹⁴C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C₃ plants.     

High-performance liquid chromatographic analysis of dipyridamole in plasma and whole blood

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of dipyridamole in plasma and whole blood. The method involves a single extraction of an alkalinized sample with diethyl ether followed by evaporation of the organic solvent and ion-pair chromatography using fluorescence detection. The lower limit of sensitivity for dipyridamole is 1 ng/ml. Concentrations of dipyridamole between 1 and 500 ng per sample are measured with an average coefficient of variation of 4.5% in plasma and 7.4% in whole blood.     

Isolation and characterization of glycolic acid oxidase from human liver.

Glycolic acid oxidase has been isolated from human liver and purified over 3000-fold to a specific activity of 123 U/mg protein by a 5-step procedure. The preparation gave a single protein band on polyacrylamide gel electrophoresis, required flavin mononucleotide for catalytic activity, had a pH optimum between 8.2-8.8 depending on the substrate, and had a molecular weight of 105 000. The enzyme has a broad specificity towards alpha-hydroxy acids. Glycolate (Km = 3.3 . 10(-4) M) was the most effective substrate. The enzyme was stable for several months when stored as an (NH4)2SO4 precipitate or in 15% glycerol. Since glycolate inhibits the oxidation of glyoxylate to oxalate by glycolic acid oxidase, it is suggested that glycolic acid oxidase contributes to the synthesis of oxalate in vivo when the glyoxylate concentration is high and the glycolate concentration is low.     

The effect of osmotic stress on the oxidation of glycolate by the blue-green alga Anacystis nidulans

Planta - Anacystis nidulans Richt. was shown to assimilate glycolic acid, and uptake was light-stimulated. In the dark 90% of the glycolate taken up was oxidised to CO2. Both light and dark uptake...