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C Brunold

Publications and source records attributed to C Brunold.

At least 19 recordsLinked to original sources
PubMed2004

Polysaccharides from Hibiscus sabdariffa flowers stimulate proliferation and differentiation of human keratinocytes.

Raw polysaccharides, previously described in detail, were isolated from the flowers of Hibiscus sabdariffa L. and fractionated by ion exchange chromatography into one neutral and three acidic subfractions. Raw polysaccharides and all acidic subfractions caused a strong induction of proliferation of human keratinocytes (HaCaT) of up to 40 %, while the neutral polymers were ineffective. While mitochondrial activity was not influenced, raw polysaccharides induced early differentiation of primary natural human keratinocytes, as determined by involucrin formation.

Cell Division↗
PubMed2004

Real-time process/quality control for HPC processing.

BACKGROUND: JACIE Standards (FACT Standards in the USA) have been implemented in Europe since 1999. An on-site accreditation inspection took place at our center in January 2004. The purpose of this work was to develop a real-time process/quality control system meeting the JACIE Standards for HPC release. METHODS: Data from 194 HPC processing procedures for autologous transplantation performed over a 5-year period were analyzed. The results of different processing methods applied at our facility were compared: (1) cryopreservation without washing cells (n=50), (2) washing cells (n=87), (3) cell-density separation (n=12) and (4) positive CD34 selection (n=45). RESULTS: Four critical control points were set for the validation of HPC processing: (a) number of lost CD34(+) cells during processing, (b) contamination, (c) viability of the cells after thawing and (d) ability to reconstitute hematopoiesis after transplantation. On the basis of statistical analysis, ranges of acceptable values were defined for each critical control point and for each processing method. Those acceptable values were used for cell release and real-time quality control. DISCUSSION: This study describes a model for the validation of HPC processing and for a real-time process/quality control system for HPC release. Optimization of processing techniques, standardization of methods and comparison between facilities will open the way towards external quality controls and quality improvement.

Antigens, CD34↗
PubMed2001

Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein.

Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of (57)Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S](2+) cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5'-phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5'-phosphosulfate reductases found in sulfate reducing bacteria.

Amino Acid Sequence↗
PubMed2001

Assimilatory sulfate reduction in C(3), C(3)-C(4), and C(4) species of Flaveria.

The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C(4) monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C(4) photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C(3), C(3)-C(4), C(4)-like, and C(4) species of the dicot genus Flaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C(4)-like and C(4) species than in C(3) and C(3)-C(4) species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C(4) species Flaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C(4) plants and therefore is neither a prerequisite nor a consequence of C(4) photosynthesis.

Asteraceae↗
PubMed2001

Increasing the glutathione content in a chilling-sensitive maize genotype using safeners increased protection against chilling-induced injury.

With the aim of analyzing their protective function against chilling-induced injury, the pools of glutathione and its precursors, cysteine (Cys) and gamma-glutamyl-Cys, were increased in the chilling-sensitive maize (Zea mays) inbred line Penjalinan using a combination of two herbicide safeners. Compared with the controls, the greatest increase in the pool size of the three thiols was detected in the shoots and roots when both safeners were applied at a concentration of 5 microM. This combination increased the relative protection from chilling from 50% to 75%. It is interesting that this increase in the total glutathione (TG) level was accompanied by a rise in glutathione reductase (GR; EC 1.6.4.2) activity. When the most effective safener combination was applied simultaneously with increasing concentrations of buthionine sulfoximine, a specific inhibitor of glutathione synthesis, the total gamma-glutamyl-Cys and TG contents and GR activity were decreased to very low levels and relative protection was lowered from 75% to 44%. During chilling, the ratio of reduced to oxidized thiols first decreased independently of the treatments, but increased again to the initial value in safener-treated seedlings after 7 d at 5 degrees C. Taking all results together resulted in a linear relationship between TG and GR and a biphasic relationship between relative protection and GR or TG, thus demonstrating the relevance of the glutathione levels in protecting maize against chilling-induced injury.

Adaptation, Physiological↗
PubMed2000

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland.

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.

Adrenal Glands↗
PubMed2000

Adenosine 5'-phosphosulfate sulfotransferase and adenosine 5'-phosphosulfate reductase are identical enzymes.

Adenosine 5'-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M(r) of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K(m) = 6.5 microM) and not adenosine 3'-phosphate 5'-phosphosulfate as sulfonyl donor. The V(max) of recombinant Lemna APS sulfotransferase (40 micromol min(-1) mg protein(-1)) was about 10 times higher than the previously published V(max) of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO(3)(2-). Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO(3)(2-) was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Amino Acid Sequence↗
PubMed2000

Inhibition of glutathione synthesis reduces chilling tolerance in maize.

The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine (gammaEC) synthetase, the first enzyme of GSH synthesis. At 25 degrees C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 degrees C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or gammaEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity.

Adaptation, Physiological↗
PubMed2000

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione.

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1. 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.

Acetyltransferases↗
PubMed2000

Sulfate assimilation in higher plants characterization of a stable intermediate in the adenosine 5'-phosphosulfate reductase reaction.

The enzyme catalysing the reduction of adenosine 5'-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 degrees C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2-3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5'-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 degrees C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2-3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2-3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

Adenosine Phosphosulfate↗
PubMed2000

Regulation of sulfate assimilation by nitrogen in Arabidopsis.

Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5'-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mM each of NO(3)(-), NH(4)(+), or glutamine (Gln), or 1 mM O-acetylserine (OAS). (35)SO(4)(2-) feeding showed that after addition of NH(4)(+), Gln, or OAS to nitrogen-starved plants, incorporation of (35)S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.

Arabidopsis↗
PubMed1999

Light regulation of assimilatory sulphate reduction in Arabidopsis thaliana.

Adenosine 5'-phosphosulphate reductase (APR) is considered to be a key enzyme of sulphate assimilation in higher plants. We analysed the diurnal fluctuations of total APR activity and protein accumulation together with the mRNA levels of three APR isoforms of Arabidopsis thaliana. The APR activity reached maximum values 4 h after light onset in both shoots and roots; the minimum activity was detected at the beginning of the night. During prolonged light, the activity remained stable and low in shoots, but followed the normal rhythm in roots. On the other hand, the activity decreased rapidly to undetectable levels within 24 h of prolonged darkness both in shoots and roots. Subsequent re-illumination restored the activity to 50% in shoots and to 20% in roots within 8 h. The mRNA levels of all three APR isoforms showed a diurnal rhythm, with a maximum at 2 h after light onset. The variation of APR2 mRNA was more prominent compared to APR1 and APR3. 35SO42- feeding experiments showed that the incorporation of 35S into reduced sulphur compounds in vivo was significantly higher in light than in the dark. A strong increase of mRNA and protein accumulation as well as enzyme activity during the last 4 h of the dark period was observed, implying that light was not the only factor involved in APR regulation. Indeed, addition of 0.5% sucrose to the nutrient solution after 38 h of darkness led to a sevenfold increase of root APR activity over 6 h. We therefore conclude that changes in sugar concentrations are also involved in APR regulation.

Arabidopsis↗
PubMed1998

Cyst(e)ine is the transport metabolite of assimilated sulfur from bundle-sheath to mesophyll cells in maize leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, gamma-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.

Journal Article↗
PubMed1994

Induction of glutathione transferase activity in wheat and pea seedlings by cadmium.

The effect of the heavy metal cadmium on the glutathione transferase (GT) activity was studied in the shoots and roots of wheat and pea seedlings. The exposure to cadmium led to the reduction of plant growth rates and to a marked induction of GT activity in both plants. In wheat the induction was stronger in the roots than in the shoots at low cadmium concentrations (40-160 microM), but at 640 microM cadmium the effect was more pronounced in the shoots (4.0-fold increase of the activity as compared to control). In pea seedlings the induction rates were generally higher in the roots than in the shoots (at 640 microM cadmium the activity in the roots was 340% of the control).

Cadmium↗
PubMed1993

Evidence for tangeretin O-demethylation by rat and human liver microsomes.

1. Tangeretin, a polymethoxylated flavone, was studied as a substrate for cytochrome P450-catalysed demethylation reactions by rat and human liver microsomes. Evidence has been presented for the production of formaldehyde in the presence of tangeretin and NAD(P)H. Kinetic studies showed a Km value for tangeretin of about 18 microM in both species. 2. The reaction was inhibited by CO, piperonyl butoxide, 7,8-benzoflavone, propyl gallate, aminobenzothiazole and metyrapone. 3. Rats pretreated with classical cytochrome P450 inducers (Aroclor 1254, 3-methylcholanthrene, phenobarbital, dexamethasone and ciprofibrate) or with flavonoids (flavone, flavanone, quercetin and tangeretin) resulted in increased microsomal demethylation of tangeretin after 3-methylcholanthrene and flavone only. Tangeretin did not enhance its own metabolism. 4. Tangeretin interacted with the oxidized form of cytochrome P450 to produce a reverse type I spectrum. 5. Results indicate that tangeretin is metabolized in liver microsomes by an O-demethylation reaction involving cytochrome P450.

Animals↗
PubMed1983

Measurement of ferredoxin-dependent sulfite reductase activity in crude extracts from leaves using O-acetyl-L-serine sulfhydrylase in a coupled assay system to measure the sulfide formed.

Ferredoxin-dependent sulfite reductase (EC 1.8.7.1) catalyses the reduction of sulfite to sulfide, using reduced ferredoxin as an electron donor. An assay system was developed for measuring this enzyme activity in crude extracts and broken chloroplast preparations from leaves. The assay consists of a coupled system in which the sulfide formed is used for cysteine synthesis by added O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8). Cysteine thus formed is determined with ninhydrin under conditions where O-acetylserine does not react and serves as a measure for ferredoxin-dependent sulfite reductase activity. Cysteine synthesized in the assay can be determined from 10 to 200 nmol. One assay per minute can be performed.

Cysteine↗
PubMed1976

Subcellular localization of O-acetylserine sulfhydrylase in spinach leaves.

A combination of differential centrifugation and isopycnic sucrose density gradient centrifugation of extracts from spinach leaves (Spinacia oleracea L.) shows that about 20% of the O-Acetylserine sulfhydrylase are associated with chloroplasts. No appreciable amounts of O-Acetylserine sulfhydrylase band with mitochondrial and peroxisomal marker enzymes.

Chlorophyll↗