Susanne Sørensen

ARABINAN DEFICIENT 1 Is a Putative Arabinosyltransferase Involved in Biosynthesis of Pectic Arabinan in Arabidopsis

The function of a putative glycosyltransferase (At2g35100) was investigated in Arabidopsis (Arabidopsis thaliana). The protein is predicted to be a type 2 membrane protein with a signal anchor. Two independent mutant lines with T-DNA insertion in the ARABINAN DEFICIENT 1 (ARAD1) gene were analyzed. The gene was shown to be expressed in all tissues but particularly in vascular tissues of leaves and stems. Analysis of cell wall polysaccharides isolated from leaves and stems showed that arabinose content was reduced to about 75% and 46%, respectively, of wild-type levels. Immunohistochemical analysis indicated a specific decrease in arabinan with no change in other pectic domains or in glycoproteins. The cellular structure of the stem was also not altered. Isolated rhamnogalacturonan I from mutant tissues contained only about 30% of the wild-type amount of arabinose, confirming the specific deficiency in arabinan. Linkage analysis showed that the small amount of arabinan present in mutant tissue was structurally similar to that of the wild type. Transformation of mutant plants with the ARAD1 gene driven by the 35S promoter led to full complementation of the phenotype, but none of the transformants had more arabinan than the wild-type level. The data suggest that ARAD1 is an arabinan α-1,5-arabinosyltransferase. To our knowledge, the identification of other l-arabinosyltransferases has not been published.

Identification of a Xylogalacturonan Xylosyltransferase Involved in Pectin Biosynthesis in Arabidopsis

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

Arabinoxylan Biosynthesis in Wheat. Characterization of Arabinosyltransferase Activity in Golgi Membranes

Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[14C]-β-l-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[14C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[14C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [14C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn2+ being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [14C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.

Chromatography and capillary electrophoresis in food analysis

General Aspects of Experimental Biochemistry Buffers and Micelles Binding, Association, Dissociation and Kinetic Extraction of Native LMW and HMW Biomolecules Spectroscopy and Detection Methods Liquid Chromatography Ion-exchange Chromatography High Performance Liquid Chromatography and Fast Polymer Liquid Chromatography Electrophoresis High Performance Capillary Electrophoresis Analytical Determination of Low Molecular Weight Compounds Protein Purification and Analysis Immunochemical Techniques Analysis of Dietary Fibre Appendix: Supercritical Fluid Extraction (SFE) and Supercritical Fluid Chromatography (SFC) Subject Index.

Supercritical fluid chromatography as a method of analysis for the determination of 4-hydroxybenzylglucosinolate degradation products.

In the present study analytical and preparative supercritical fluid chromatography (SFC) were used for investigation of myrosinase catalysed degradation of 4-hydroxybenzylglucosinolate (sinalbin). Sinalbin occurs as a major glucosinolate in seeds of Sinapis alba L., in various mustards and other food products. The degradation products were identified and quantified by analysis based on a developed SFC method using a bare silica column. Determinations comprised transformation products of sinalbin, produced both during degradation of isolated sinalbin, and during autolysis of meal from S. alba seeds. The conditions in the developed SFC method were used as basis for the preparative SFC procedure applied for isolation of the components prior to their identification by nuclear magnetic resonance (NMR) spectroscopy. Myrosinase catalysed sinalbin hydrolysis resulted in the reactive 4-hydroxybenzyl isothiocyanate as an initial product at pH values from 3.5 to 7.5 whereas 4-hydroxybenzyl cyanide was one of the major products at low pH values. 4-Hydroxybenzyl isothiocyanate was found to disappear from the aqueous reaction mixtures in a few hours, as it reacted easily with available nucleophilic reagents. 4-Hydroxybenzyl alcohol was found as the product from reaction with water, and with ascorbic acid, 4-hydroxybenzylascorbigen was produced.

Determination of vitamins in food based on supercritical fluid extraction prior to micellar electrokinetic capillary chromatographic analyses of individual vitamins

The separation of 14 water-soluble vitamins and vitamin cofactors was investigated by micellar electrokinetic capillary chromatography and diode array detection using sodium cholate as the micellar phase. The method was optimised with respect to the effect of buffer composition, capillary temperature and applied voltage resulting in separation of all compounds in about 25 min. With the current method it is possible to predict the eluting order of the individual compounds from their net charges of each compound because of a low ion pairing between solutes and micelles. The linearities within concentration ranges of up to two-orders of magnitudes were good with correlation coefficients from 0.971 to 0.997. The separation efficiency was satisfactory with a good resolution ranging from 2 to 45 and a theoretical number of plates varying from 200,000 to 480,000. The repeatability of the developed method showed relative standard deviations on migration time in the range from 0.5% to 1.2% (n = 15) and for normalised peak areas, relative standard deviations were approximately 6%.

Supercritical fluid chromatography as basis for identification and quantitative determination of indol-3-ylmethyl oligomers and ascorbigens.

Indol-3-ylmethylglucosinolate (glucobrassicin) occurs in most plants of the Brassicaceae family together with hydroxy and methoxy derivatives of glucobrassicin. These compounds and products produced therefrom have been the subject of considerable research interest due to their potential anticarcinogenic effects, and thereby a need for techniques to work with the individual compounds. A method using normal-phase supercritical fluid chromatography (SFC) with methanol as modifier has been developed for determination and quantification of the various indol-3-ylmethyl derivatives including ascorbigens formed from the glucobrassicin degradation product, indol-3-ylmethanol, under acidic conditions (pH 2-6) with and without the presence of ascorbic acid. The SFC method had detection limits in the 10-100-pmol range. In the absence of ascorbic acid a range of oligomers were formed, whereas the presence of ascorbic acid favoured the formation of ascorbigen and products thereof. Quantitatively important indol-3-ylmethyl oligomers consisting of up to five indol rings have been purified with preparative SFC and identified from MS and 1D and 2D NMR experiments with complete assignment of chemical shifts to all of the atoms. Investigation of the autolysis products of white cabbage showed that ascorbigens were the quantitatively dominating degradation products of indol-3-ylmethylglucosinolates.

Subcellular Distribution of Urokinase and Urokinase Receptor in Human Neutrophils Determined by Immunoelectron Microscopy

A high-affinity receptor for urokinase-type plasminogen activator (uPAR) has been identified on the plasmamembrane of a number of different cell types, and has been shown to be important for plasminogen activation, cell adhesion, and possibly signal transduction. uPAR and uPA cosediment with secretory vesicles and specific granules by subcellular fractionation and translocate to the plasma membrane upon activation of neutrophils. Here the subcellular distribution of uPAR and uPA is studied by electron microscopy of neutrophils using immunogold double labeling for uPAR and uPA and a set of markers for well-defined subtypes of granules: matrix metalloproteinase type-9 (MMP-9) for gelatinase granules, lactoferrin (LF) for specific granules, and myeloperoxidase (MPO) and neutrophil elastase (NE) for primary granules. With this technique uPAR colocalizes with uPA in 71% of labeled granules. In granules containing uPAR the degree of coexpression with MMP-9, MPO and NE was 19, 66, and 74%, respectively. In granules labeled for uPA the corresponding overlap with MMP-9, MPO and NE was 24, 64, and 51%, respectively. Low levels of co-localization were found for uPAR and LF (7%) and for uPA and lactoferrin (5%). The results indicate that uPAR and uPA arepresent in gelatinase granules and primary granules, but rarely in specific granules. The demonstration of uPAR and uPA in primary granules is of particular interest, and may indicate that uPAR and uPA participate in the activation of latent hepatocyte growth factor of neutrophils.

Subcellular Localization of Intercellular Adhesion Molecule-1 in Colonic Mucosa in Ulcerative Colitis

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells. In ulcerative colitis (UC), ICAM-1 is suggested also to be involved in the further migration of leukocytes toward the epithelial lining, and in colonic tissue it has been reported to be expressed by cell types other than endothelial cells. This study aimed at determining the ultrastructural localization of ICAM-1 on cells belonging to the colonic mucosa from patients with UC. Colonic biopsies from 3 UC patients and 3 control subjects were examined ultrastructurally by immunogold labeling of ICAM-1. ICAM-1 was expressed on the luminal cell membranes of endothelial cells in both controls and inflamed and noninflamed UC colon, although the density was significantly increased in UC ( p <. 0001). Labeling was observed on the basal endothelial cell membranes and on macrophages and plasma cells in inflamed UC colon only. Epithelial cells did not express ICAM-1. ICAM-1 appears to be constitutively upregulated on the luminal endothelial membrane in UC, and the expression on basal endothelial membranes in active UC only suggests that ICAM-1 is more extensively involved in the leukocyte migration than previously acknowledged.

Effects of novel processing techniques on glucosinolates and membrane associated myrosinases in broccoli.

Abstract High pressure/high temperature (HP/HT) and pulsed electric field (PEF) treatment of food are among the novel processing techniques considered as alternatives to conventional thermal food processing. Introduction of new processing techniques with fast and gentle processing steps may reveal new possibilities for preservation of healthy bioactive compounds in processed food. However, effects on various food components due to autolysis and fast reactions prior to the applied HP/HT or PEF need to be considered as the total contribution of processing steps affects the obtained food quality. The present experiments were performed on broccoli (Brassica oleracea var. Italica) florets, purée and juice. Specific focus was given to effects of HP/HT and PEF processing on the content of glucosinolates and activities of myrosinase isoenzymes (EC.3.2.1.147) in the broccoli preparations. Certain conditions applied in HP/HT processing of broccoli florets were able to maintain a high level of intact glucosinolates. Treatment at 700 MPa and 20°C for 10 min was found to inactivate myrosinase activity, but also pressure treatments at 300 MPa and 20°C were able to maintain a high level of intact glucosinolates present in the untreated broccoli florets. PEF processing of broccoli purée and juice showed that the myrosinase activities resulted in nearly total glucosinolate transformations as result of autolysis during puréeing and juice making prior to the PEF processing. These data demonstrated that insight into potential effects on myrosinase activities from application of PEF processing implies specific focus on the sample steps preceding the PEF processing