Amounts Of Galactose Research Articles

Evaluation of Chemical and Morphological Properties of Spruce Wood Stored in the Natural Environment.

This paper focuses on the changes in chemical structure and fiber morphological properties of spruce wood during 15 months of its storage in an open forest woodshed. From the chemical composition, the extractives, cellulose, holocellulose, and lignin content were determined. The pH value was measured on the wood surface using a contact electrode. Acetic and formic acid, saccharides (glucose, xylose, galactose, arabinose and mannose), and polymerization degree (PD) of cellulose were analyzed using the HPLC method. Fiber length and width were determined using a fiber tester analyzer. After 15 months of storage the content of both cellulose (determined by the Seifert method) and lignin did not change; the quantity of hemicelluloses decreased by 13.2%, due to its easier degradation and less stability compared to cellulose; and the pH value dropped by one degree. HPLC analyses showed a total decrease in the cellulose DP of 9.2% and in saccharides of 40.2%, while the largest decreases were recorded in the quantity of arabinose, by 72%, in the quantity of galactose, by 61%, and in the quantity of xylose, by 43%. Organic acids were not detected due to their high volatility during wood storage. The total decrease in average fiber length was 38.2% and in width was 4.8%. An increase in the proportion of shorter fibers, and a decrease in the proportion of longer fibers, was recorded. It can be concluded that fundamental changes occurred in the wood, which could affect the quality of further products (e.g., chips, pulp, paper, particleboards).

Research of Inonotus obliquus Oligosaccharide in Prevention of Hyperlipidemia

In this study, hot water was used to extract Inonotus obliquus oligosaccharide. DEAE-cellulose and Sepharose G-200 were used to purify Inonotus obliquus oligosaccharide. Inonotus obliquus oligosaccharide IOP-2A was obtained. Its molecular weight Mw is about 1000 Da. The monosaccharide composition and molar ratio were glucose : xylose : galactose : mannose = 54.1 : 13.6 : 13.2 : 6.7. In addition, it also contains a small amount of galactose, gluconic acid, rhamnose, and fucose. IOP-2A contained mainly β-glycosidic bonds. Among them, 1,4-glycosidic bonds accounted for 9.2%, and 1,6-glycosidic bonds accounted for 85.1%. Oligosaccharide macromolecules formed a layered structure. Mouse experiments showed that IOP-2A had the function of preventing hyperlipidemia. At the same time, IOP-2A had a certain protective effect on the liver and kidney. The mechanism of IOP-2A in preventing hyperlipidemia was obtained from the perspective of mouse intestinal flora.

Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening.

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.

Effect of temperature on the equilibrium and kinetics of galactose, glucose, and lactose adsorption on a cation exchanger

AbstractGalacto-oligosaccharides are typically produced by an enzymatic reaction when the post-reaction mixture contains considerable amounts of lactose and glucose and a smaller amount of galactose. In order to develop a process of chromatographic removal of saccharide impurities, adsorption equilibria and kinetics of these di- and monosaccharides were investigated for Diaion UBK 530, an industrialgrade strong cation-exchanger in the Na+ form. Frontal chromatographic experiments were carried out in the temperature range of 30–70°C and a broad interval of saccharide concentrations up to 350 g L−1. Breakthrough curves were described using the equilibrium-dispersive model with the linear adsorption isotherm. Both the distribution and the axial dispersion coefficient values depended on the saccharide molecule type and size. No significant effect of temperature or concentration on the distribution coefficient was observed. The apparent dispersion coefficients of all saccharides exhibited some decrease with the temperature, which was caused by the decrease of the intraparticle mass transfer resistance. An analysis showed that both the intraparticle mass transfer and the axial dispersion had a significant influence on the front dispersion.

Electro-Catalytic Production of Lactulose: Statistical Modeling and Experimental Validation

<p>Full factorial experimental design was used to optimize the operating conditions of lactose converting into lactulose under electro-activation conditions. The ANOVA showed a high correlation coefficient R<sup>2</sup> (0.9889) and a low root mean square error (1.4 ± 0.15%) values for the regression model of the experimental design, indicating the good predictive nature of the model. The obtained results after optimisation showed that the optimum electro-isomerization conditions are the electric current of 300 mA, temperature of 10 °C, and reactor configuration in which a cation exchange membrane separated the central compartment from the cathodic one in which the electro-isomerization reaction was conducted. According to the optimized conditions, the maximum lactose conversion into lactulose was 30 ± 1.23%. As by-products of the electro-activation, only small amounts of galactose were observed.</p>

Carbohydrates from Solidago caucasica herb

Solidago caucasica Kem.-Nath. is a goldenrod of the family Asteraceae and is an unstudied species endemic to the Caucasus although other species of the genus Solidago are widely used worldwide to treat diseases of the urogenital system. We previously detected in S. caucasica herb flavonoids (rutin, vicenin, hesperidin), coumarins (umbelliferone, esculetin, dihydrocoumarin), phenolcarboxylic acids (gallic, chicoric, chlorogenic, and caffeic acids) [1], organic acids (citric, malic, and succinic) [2], 15 amino acids, 5 macroelements, and 16 microelements [3]. We studied the carbohydrate composition of S. caucasica herb collected in subalpine meadows of the western Caucasus (Daut Gorge of Karachay-Cherkessia Republic). Raw material (30.0 g of S. caucasica herb) was extracted with CHCl 3 (2 100 mL) and dried. The remaining raw material was extracted twice with refluxing EtOH (82°C) in order to isolate sugars soluble in alcohol (SSA). The resulting extracts were evaporated and chromatographed on Filtrak FN 7,12 paper (PC) using BuOH–Py–H 2 O (6:4:3). SSA according to PC were glucose, galactose (anilinium biphthalate detector), fructose, and sucrose (5% urea detector). Next, polysaccharides were isolated by successive extraction of water-soluble polysaccharides (WSPS) with H 2 O, pectinic substances (PS) with oxalic acid and ammonium oxalate solutions (0.5%), and hemicellulose (HMC) with base solution (5%). The monosaccharide composition of the carbohydrates was established by acid hydrolysis [4]. Neutral sugars were identified by GC. Samples were analyzed by GC on a Chrom-5 chromatograph with a flame-ionization detector, glass column (1.5 m 0.3 m) with 5% Silicone XE-60 on Chromaton NAW (0.200–0.250 mesh), 210°C, He carrier gas at 30 mL/min, and aldononitrile acetates [4]. Table 1 presents the results. The isolated WSPS and PS were assigned according to the amount of component monosaccharides as galactans; HMC, xylans. The greatest amount of galactose was observed in PS fractions. The PS and HMC fractions were the main ones with respect to content. Uronic acids were identified in all fractions. The amount of uronic acids was determined by a photoelectrocolorimetric method using the reaction with carbazole in H 2 SO 4 solution [5]. The greatest amount of uronic acids was detected in PS. The accumulation of these components was responsible for the prospective pharmaceutical use of the carbohydrates. WSPS were a dark-brown powder with relative viscosity 3.0 (c 1%, H 2 O). The content of free carboxylic acids (Cf) was established by titration as 1.98%; of esterified carboxylic acids (Ce), 4.6%; the degree of esterification (), 69.9%. Therefore, WSPS were highly esterified compounds. This allowed them to be recommended for use as excipients for manufacturing drug forms as gel-formers, thickeners, stabilizers, etc. PS were a dark-brown powder that dissolved in H 2 O with heating to form viscous solutions of relative viscosity 10.17 (c 1%, H 2 O). The content of Cf was 6.75%; Ce, 3.78%; 35.89%. Therefore, PS were low-esterified compounds. The rather high content of free carboxylic acids made this plant a promising subject for isolating PS with pronounced sorption properties, especially with respect to metal ions. HMC was a friable light-brown powder that dissolved in base. IR spectra of samples were taken in pressed KBr pellets on a Model 2000 IR-Fourier spectrometer (PerkinElmer) using 100 scans. The following conclusion could be made based on an analysis of the found characteristic IR absorption bands of the carbohydrates.

Studies on the Preliminary Characterization of a Novel Exopolysaccharide Produced by <i>Streptococcus thermophilus </i>Strain from Tibetan Kefir Grain

The novel exopolysaccharide (EPS) was produced by a streptococcus thermophilus strain isolated from Tibetan kefir grain, and it was purified using DEAE cellulose 52 and DEAE Sepharose CL-6B column chromatography. Then it was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular mass was estimated to be 30,092Da. Infrared spectrum analysis indicated it had characteristic glycosidic structures. Gas chromatography showed that the EPS is composed of mannose and glucose in a molar ratio of 2.6:1 with trace galactose. The structural features of EPS were investigated by partial hydrolysis with acid, methylation analysis and NMR spectroscopy analysis. The results revealed that the main backbone chain of EPS was (23)-β-D-Manp, (34)-β-D-Manp, (23,6)-α-D-Glcp and trace amounts of galactose.

Feeding rats a high fat/carbohydrate ratio diet reduces jejunal S/I activity ratio and unsialylated galactose on glycosylated chain of S–I complex

Aims We examined whether decreasing jejunal sucrase/isomaltase (S/I) activity ratio by feeding rats a high fat/carbohydrate ratio diet is regulated by changing glycosylated chains on the S–I complex. Main methods Jejunal activities of sucrase, isomaltase and β-1,4-galactosyltransferase were examined in rats fed a high fat/carbohydrate or a low fat/carbohydrate ratio diet. The amount of galactose and mannose in the glycosylated chain on the S–I complex in rats fed both diets was determined using RCA 120 and Con A lectins, respectively. The effects of reducing unsialylated galactose from the glycosylated chain on the S–I complex were assessed by determining sucrase activity in purified S–I complex treated with β-galactosidase. Key findings and significance Feeding rats a high fat/carbohydrate ratio diet reduced jejunal S/I activity ratio in mucosal homogenates and purified fractions. The level of unsialylated galactose in glycosylated chains on the S–I complex was reduced by feeding rats a high fat/carbohydrate ratio diet. The form with reduced levels of unsialylated galactose had lower sucrase activity than that with more unsialylated galactose. The reduction of galactose on the S–I complex by β-galactosidase in vitro reduced sucrase activity. Feeding rats a high fat/carbohydrate ratio diet also reduced jejunal β-1,4-galactosyltransferase activity. Taken together, decreasing the S/I activity ratio by feeding rats a high fat/carbohydrate diet is associated with the reduction of unsialylated galactose on the glycosylated chain of the S–I complex.

Quantitative chemical indicators to assess the gradation of compression wood

Abstract A chemistry-based parameter has been sought for determining the gradation of compression wood (CW), i.e., the severity, in tissues of Pinus radiata wood. Fluorescence microscopy was the reference for characterisation of the tissues containing CW. The collected material contained CW of varying severity, beginning with normal wood (NW containing no CW), continuing with material with some features of CW (CW of mild severity, MCW) and ending up with a material with pronounced features of CW (CW of high severity, SCW). Matching opposite wood (OW) was also included in the study. The chemical analyses included lignin determination, sugar analysis in the acid hydrolysate, thioacidolysis, 31P-NMR spectroscopic analysis and steric exclusion chromatography of thioacidolysis products. As the severity of CW changed progressively from NW through MCW to SCW, all chemical parameters changed concurrently. In particular, levels of galactose and lignin increased, while those of glucose and mannose decreased. The amounts of p-hydroxyphenyl β-ethers released by chemical degradation and uncondensed p-hydroxyphenyl C-9 units also increased at elevated CW severity levels. The amounts of galactose and the p-hydroxyphenyl content of the lignin correlated linearly with lignin for CW samples. The chemical differences between CW and OW in the stem, branch and seedling were similar, i.e., they are independent of the morphological origin of the sample. Parameters based on the p-hydroxyphenyl unit content appear the most suitable chemical indicators of CW severity, as they are least sensitive to the sample's morphological origin and their response to CW severity is high.

Alterações nas atividades das enzimas alfa-galactosidase e poligalacturonase e nas reservas de carboidratos de sementes de Schizolobium parahyba (vell.) Blake (guapuruvú) durante a germinação

A retomada do metabolismo do embrião durante a germinação é realizada por processos metabólicos que culminam na protrusão da radícula e no fornecimento de energia para o desenvolvimento inicial da plântula. O objetivo deste trabalho foi estudar as variações nas atividades das enzimas alfa-galactosidase e poligalacturonase e nas reservas de mono e oligossacarídeos em sementes de guapuruvú durante a germinação. Para tanto, as sementes foram colocadas para germinar e as reservas do eixo embrionário e cotilédones, avaliadas periodicamente. Os teores de galactose no eixo embrionário diferiram significativamente somente entre a testemunha e o oitavo dia, muito embora houvesse aumento contínuo até o quarto dia. Somente no sexto dia de germinação houve aumento no teor de galactose nos cotilédones. Houve tendência de aumento nos teores de arabinose, manose e glicose no eixo embrionário, não sendo detectada a presença de xilose no oitavo dia. Nos cotilédones os mesmos açúcares não foram originalmente detectados no tempo zero, mas apresentaram valores mais altos nas amostras do oitavo dia. Os teores de galactose oscilam tanto no eixo embrionário, quanto nos cotilédones durante o período de germinação de sementes de guapuruvu. Os teores de sacarose aumentam e os de rafinose decrescem nos cotilédones e no eixo embrionário. Os teores de estaquiose permanecem aproximadamente estáveis no eixo e nos cotilédones, com decréscimo no eixo, no oitavo dia. A enzima alfa-galactosidase é pré-formada, tendo clara redução na sua atividade específica, no segundo dia, permanecendo constante até o oitavo. A atividade nos cotilédones apresenta aumento no quarto dia, decrescendo posteriormente. A enzima polygalacturonase é tambem pré-formada, com maior atividade inicial no eixo. A atividade nos cotilédones aumenta até o sexto dia, alcançando maiores valores que o do eixo embrionário e, em seguida, decresce para valores menores do que aqueles.

Interaction between Proteins and Polyphosphazene Derivatives Having a Galactose Moiety

For tissue engineering applications, it is necessary to balance the need for specific biological interactions with the need to prevent unfavorable nonspecific interactions. For this purpose, novel poly[(organo)phosphazenes] were synthesized having galactose and/or poly(ethylene glycol) (PEG) side chains. The synthesis was described previously. Here, we investigate the human serum albumin (HSA) adhesion to these polymers using surface plasmon resonance (SPR). We could conclude that the incorporation of PEG reduced the protein adsorption. The influence of the galactose moieties was investigated using SPR and a sugar-lectin binding assay. The interaction between a lectin (Peanut agglutinin, PNA or Ricinus communis-agglutinin, RCA) and the polyphosphazene derivatives was evaluated. Type IIA polymers, having aminohexyl-galactose, phenylalanine ethyl ester, and glycine ethyl ester side chains, were capable of binding with the lectin. As the amount of galactose was increased, the extent of the galactose specific lectin binding was also increased (higher RU or absorbance). PEG containing polymers failed to bind specifically with the lectin. The presence of PEG, either as a spacer or as additional chains, interfered with the establishment of contact between the galactose and the binding site on the lectin. The adsorption of PNA or RCA to these types of polymers was attributed to nonspecific interactions. SPR was also used to determine rate and equilibrium constants. In addition the effect of the addition of water soluble polyphosphazenes on the enzymatic cleavage of o-nitrophenyl-beta-D-galactopyranoside was investigated. The galactose moieties were not available as inhibitors because of the presence of PEG.

Study of antioxidant activities of sulfated polysaccharides from Laminaria japonica

The composition, molecular weight and in vitro antioxidant activity of various sulfated polysaccharides obtained by anion exchange chromatography, acid hydrolysis and radical process degradation of the crude sulfated polysaccharide extracted from Laminaria japonica were compared. The low sulfated F-A2, with a peak-molecular weight (Mp) of 5–15 kDa, 14.5% sulfated ester and 21.8% glucuronic acid, exhibited a very strong antioxidant activity on superoxide and hydroxyl radicals, with activity even higher than that of large molecular weight fractions F-A and F-B. However, highly sulfated fractions with a peak-molecular weight below 15 kDa had much lower antioxidant activities than other fractions. These results indicated that the sulfate group of the low molecular weight fractions represents a physical block for the reaction with oxygen radicals. The chemical properties and antioxidant activities of sulfated polysaccharide fractions obtained by radical process degradation of crude sulfated polysaccharide were quite different from those obtained by acid hydrolysates. By radical process degradation, the high molecular weight was decreased to give LM2 (Mp 8 kDa) and LM1 (Mp 1.5 kDa), with a yield of 40% and 15%, respectively. LM2 was enriched with fucose and sulfated ester, while containing low amounts of glucuronic acid. The antioxidant activity showed that LM2 was unable to scavenge either superoxide or hydroxyl radical, which suggested that radical process degradation targeted mainly ascopyllan-like species rich in glucuronic acid, while the fraction rich in sulfated l-fucose remained unchanged. However, LM1 with Mp 1.5 kDa still retained apparent scavenging ability for superoxide radical, although it contained no glucuronic acid and certain amounts of galactose and mannose as main neutral sugars. These result suggest that the antioxidant activity of sulfated polysaccharides is apparently related not only to molecular weight and sulfated ester content, as previously determined, but also to glucuronic acid and fucose content.

The chemistry of carcin oembryonic antigen

The structure of carcinoembryonic antigen is being determined as an approach to greater diagnostic specificity in the radioimmune assay. Examination by combined gas chromatography-mass spectrometry of derivatized saccharides from CEA has given a general outline of its carbohydrate structure. Sialic acid and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate oxidation (Smith degradation) destroyed additional amounts of galactose and mannose, as well as significant amounts of N-acetylglucosamine. Antigenic activity persisted, indicating that the residues destroyed played little, if any, part in the antigenicity of DEA.

Influence of processing on the content of sugars in green Arabica coffee beans

Quantitative analyses of low molecular sugars in green coffees (Coffea arabica L. var. Acaia) that were processed in parallel either by the traditional wet or the traditional dry method, revealed a close correlation between the kind of post-harvest treatment and the contents of fructose and glucose. While in washed coffee beans (wet processing) only low amounts of these both hexoses were present, those in unwashed coffees (dry processing) were significantly higher. Model-processing experiments in the laboratory confirmed these findings. Moreover, a comparison with the untreated controls revealed that the low levels of both sugars are the consequence of a decrease in the case of wet processing, whereas they remained unchanged or even increased in the case of dry processing. Further minor sugars are also affected by post-harvest treatment. The amounts of galactose, arabinose and mannose show a similar arrangement as those for glucose and fructose, although their overall concentration is about 100-fold lower. Sucrose, the major low molecular sugar in green coffee beans, is not significantly affected by coffee processing. The influence of an active seed metabolism on the observed alterations of the sugar concentrations is discussed.

Effect of fucoidans on the developing embryos of the sea urchin Strongylocentrotus intermedius

Effect of fucoidans from the Far Eastern brown algae Laminaria japonica, L. cichoroides, and Fucus evanescens on developing embryos of the sea urchin Strongylocentrotus intermedius was studied. Fucoidans from these algae were shown to have different (immunostimulating, inhibitory) effects on the developing embryos. All studied fucoidan fractions added at the zygote stage had a stimulatory effect (acceleration of developmental stages and an increase of life spans) on the developing embryos, with differences by their effective concentrations. Many fucoidans added at the late blastula stage did not produce the stimulatory effect. The lowest number of immunostimulators (only a half of the tested substances) were present among fucoidans from L. japonica cichoriodes. The best immunostimulators that increased 2–3 times the life span of the embryos were heteropolysaccharides containing mannose or xylose apart from L-fucose. Some fucoidans at high concentrations produced an inhibitory effect; they were mainly composed of L-fucose or their monosaccharide composition included, apart from fucose, a significant amount of galactose, but no mannose. The method that we used turned out to be sensitive to structural peculiarities of charged polysaccharides, so it most likely can be applied for evaluation of their immunostimulating properties. From two variants of the experiment the most sensitive was the second one, in which polysaccharides were added at the late blastula stage.

The Edwardsiella ictaluri O polysaccharide biosynthesis gene cluster and the role of O polysaccharide in resistance to normal catfish serum and catfish neutrophils

Edwardsiella ictaluri, the causative agent of enteric septicaemia of catfish (ESC), expresses long O polysaccharide (OPS) chains on its surface. The authors previously reported the construction of an isogenic Ed. ictaluri OPS mutant strain and demonstrated that this strain is avirulent in channel catfish. This paper reports the cloning of the Ed. ictaluri OPS biosynthesis gene cluster and identification of the mutated gene in the OPS-negative strain. The sequenced region contains eight complete ORFs and one incomplete ORF encoding LPS biosynthesis enzymes. The mutated gene (designated wbiT) was similar to other bacterial galactose-4-epimerases. Glycosyl composition analysis indicated that wild-type Ed. ictaluri OPS contains higher amounts of galactose and N-acetylgalactosamine than the OPS mutant strain, which correlated well with predicted functions of the genes identified in the OPS biosynthesis cluster. The OPS mutant had a relatively small, but significant, decrease in its ability to survive in normal catfish serum compared to wild-type Ed. ictaluri, but it retained the ability to resist killing by catfish neutrophils.

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture.

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.

Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms.

The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor-IgG (TNFR-IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR-IgG is a chimeric IgG fusion protein bearing one N-linked glycosylation site in the Fc region and three complex-type N-glycans in the TNF-receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH(4)Cl. The amount of galactose (Gal) and N-acetylneuraminic acid (NANA) residues on TNFR-IgG correlated in a dose-dependent manner with the ammonium concentration under which the N-linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable beta-galactosidase and sialidase activity, which increased throughout the culture. The beta-galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N-glycans was observed in incubation studies using beta-galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans-Golgi) as well as intracellular nucleotide sugar pools (increases UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). Ammonium might also affect the expression rates of beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP-GNAc pool [sum of UDP-GlcNAc and UDP-GalNAc]). The ammonium effect on TNFR-IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N-glycosylation were found in the product synthesized in the presence of glucosamine. No differences in beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH(4)Cl. pH titration of endogenous CHO alpha2,3-ST and beta-1,4-GT revealed a sharp optimum at pH 6.5, the reported trans-Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans-Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR-IgG by a pH-mediated effect on glycosyltransferase activity.

Characterisation and modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B40

The chemical structure of an exopolysaccharide produced by Lactococcus lactis subsp. cremoris B40 is studied, explaining earlier reported analytical discrepancies. The EPS was found to have a molecular mass of 6.8×10 5 g mol −1 and a molar ratio of rhamnose:galactose:glucose:phosphorus of 1:1.3:2:1.1. 31 P NMR indicated that a single phosphate group is present as a phospho diester. EPS B40 was chemically modified using 0.6 M H 2SO 4, 28 M HF or 2 M NaOH. From these modifications it could be concluded that galactose 1-phosphate was linked at the 3-position of 1,2,3,4-linked galactose in the backbone of the EPS. Furthermore, it appeared that during the hydrolysis step of the sugar composition analysis the galactose 3-phosphate linkages were only partially split and that, as a result, the amount of galactose was underestimated in presence of phosphate. Finally, it was demonstrated that a crude cellulase preparation was able to degrade dephosphorylated and partially de-rhamnosylated EPS.

The role of UDP-glucose epimerase in carbohydrate metabolism of Arabidopsis.

Uridine 5'-diphospho-glucose-4-epimerase (UDP-Glc epimerase) catalyses the reversible epimerization of UDP-galactose and UDP-glucose. In contrast to bacteria and yeast, expression of the UDP-Glc epimerase gene in Arabidopsis was found not to be induced by galactose. To elucidate the metabolic role of this enzyme, transgenic Arabidopsis plants expressing the respective cDNA in sense or antisense orientation were constructed, leading to a range of plant lines with different UDP-Glc epimerase activities. No alterations in morphology were observed and the relative amounts of different galactose-containing compounds were not affected if the plants were raised on soil. However, on agar plates in the presence of galactose, the growth of different lines was increasingly repressed with decreasing enzyme activity, and an increase in the UDP-Gal content was observed in parallel, whereas the UDP-Glc content was nearly constant. The amount of galactose in the cell wall was increased in plants with low UDP-Glc epimerase activity grown on galactose, whereas the cellulose content in the leaves was not altered. Furthermore, starch determined at different times of the day was highly abundant in plants with low UDP-Glc epimerase activity in the presence of galactose. It is proposed that low endogenous UDP-Glc epimerase activity is responsible for the galactose toxicity of the wild-type. Possible mechanisms by which the starch content might be modulated are discussed.