Superoxide dismutase (SOD) was first isolated by Mann and Keilis (1938) and its catalytic function, which consists to dismutate O2- into molecular oxygen and hydrogen peroxide, was discovered in 1969 by McCord and Fridovich [9]. Mammals have two forms of SOD isozymes: the manganese SOD (Mn-SOD), present in the mitochondria, and the copper/zinc SOD (Cu/Zn-SO), present in

the cytoplasm [10, 11]. In plants, SOD have been classified into three distinct types on the basis of their metal cofactor: Cu/Zn-SOD (in the cytosol and chloroplasts), Mn-SOD (in mitochondria), and Fe-SOD (often in chloroplasts) [12–14]. There are three known SOD in E. coli: MnSOD, FeSOD MLN2238 and CuZnSOD. The two first are located in the cytoplasm and the last in the periplasmic space [15]. A distinct additional fourth class of SOD containing nickel PLX4032 price (NiSOD) was recently discovered in Streptomyces

[16, 17] and cyanobacteria [18]. SOD-driven dismutation was the only biological mechanism identified for scavenging superoxide anion radicals until the early 1990′s. McCord et al. [19] established a correlation between oxygen tolerance and SOD production and suggested that SOD was the single most important enzyme for enabling organisms to survive in the presence of molecular oxygen. They proposed that the hypersensitivity of obligate anaerobes to oxygen was a consequence of SOD deficiency. However, most anaerobic organisms, which indeed lack SOD, show various degrees of tolerance to oxygen when they are occasionally exposed

to this molecule in their environments. Two novel iron-sulphur-containing proteins that detoxify superoxide molecules were then discovered in sulphate-reducing and hyperthermophilic anaerobes: desulfoferrodoxin (Dfx) in Desulfovibrio desulfuricans, Desulfovibrio vulgaris Hildenbourgh [20] and Desulfoarculus baarsii [21], neelaredoxin (Nlr) in Desulfovibrio gigas [22] and superoxide reductase (SOR) in Pyrococcus furiosus [23]. This revealed the existence of alternative mechanisms for ROS detoxification in anaerobes. The Sitaxentan function of these proteins was first studied in 1996 by Dfx complementation of superoxide detoxication activity in E. coli SOD mutants [24]. Later, Nlr from Treponema pallidum [25] and D. gigas [26] were also shown to complement such SOD mutants. Liochev and Fridovich [27] suggested that Dfx catalyzes the reduction of superoxide rather than its dismutation, and that it uses cellular reductants such as NAD(P)H. Subsequently, the Dfx enzyme was confirmed as an oxidoreductase [23–25, 27]. Finally, the superoxide reductase activity of those proteins were established by two groups [21, 23]. Dfx and Nlr proteins have different numbers of iron sites: both contain a similar C-terminal single iron-containing site (click here centre II) but also has Dfx a second N-terminal site (centre I) [22, 28].

Therefore, it can be suggested that in the given systems, the combustion see more process in the K2TaF7 + (5 + k)NaN3 + kNH4F system starts at around 350 ± 50°C. Figure 3 DSC-TGA curves of K 2 TaF 7 + 5NaN 3 and K 2 TaF 7 + 9NaN 3 + 4NH 4 F systems in argon atmosphere. Figure 4 shows the

this website temperature-time profiles for the combustion wave of the K2TaF7 + (5 + k)NaN3 + kNH4F mixture over the reaction time (t). As shown in Figure 4, the starting temperature for the combustion process is denoted by T* (350 ± 50°C) and corresponds to the sharp peaks in the DSC curve (Figure 3). One can see that in the beginning of the reaction zone, the temperature increases rapidly from 25°C to 700°C and then to 1,000°C, and then long-tailed post-combustion processes followed. The combustion temperature (T c) showed a tendency to decrease Tozasertib in vitro with the amount of NH4F used. In the investigated interval of k, the T c drops from 1,170°C (k = 0) to 850°C (k = 4). The maximum combustion velocity (U c = 0.5 cm/s) occurred at the nearly stoichiometric mixture (k = 0), but combustion velocity decreased significantly as the K2TaF7 + 5NaN3 mixture became ‘diluted’ with NH4F. Figure 4 Temperature-time profiles in K 2 TaF 7 + (5 + k )NaN 3 + k NH 4 F system. Characteristics of combusted samples and powders Figure 5 shows photographs of the as-combusted (Figure 5a,b) and water-purified (Figure 5c) samples. After combustion,

the sample of the K2TaF7 + 5NaN3 composition (k = 0) retained its original shape and size (Figure 5a). However, the samples produced using 2.0 to 4.0 mol of NH4F had melted after the combustion process, forming a brown-colored, brittle, and shapeless molten product. For instance, several fragments of the sample prepared with k = 4 are shown in Figure 5b.

Many large pores, due to the release of N2 and H2 gases during the combustion process, can be seen in the solid molten mass. After dissolving alkali fluorides (NaF and KF) into warm distillated water, TaN fine powders were obtained. A photograph of finally purified TaN samples prepared from the K2TaF7 + 5NaN3 Olopatadine +4NH4F mixture is shown in Figure 5c. Its color is uniformly black, and specific gravity lies between 0.7 and 0.9 g/cm3. Figure 5 Photographs of as-combusted (a, b) and water-purified (c) samples. The XRD patterns for the water-purified powders that had been prepared with different amounts of NH4F are shown in Figure 6. Diffraction peaks of the sample prepared at k = 0 (without NH4F) indicate three nitride phases: hexagonal ε-TaN, TaN0.8, and Ta2N (Figure 6a). The cubic δ-TaN phase was detected in large amounts, along with the ε-TaN and TaN0.8 phases for samples with k = 2 (Figure 6b). By applying 4 mol of NH4F to the reaction of K2TaF7 and NaN3, the only crystalline product produced is cubic TaN. The diffraction peaks marked in Figure 6c correspond to face-centered cubic TaN (JCPDS 32–1283).

Int J Food Microbiol 2006, 108:125–129.PubMedCrossRef 30. Liao LF, Lien CF, Lin JL: FTIR study of adsorption and photoreactions of acetic acid on TiO2. Phys Chem Chem Phys 2001, 3:831–837.CrossRef 31. Jackson M, Ramjiawan B, Hewko M, Mantsch

HH: Infrared microscopic functional group mapping and spectral clustering analysis of hypercholesterolemic rabbit liver. Cell Mol Biol 1998, 44:89–98.PubMed 32. Nichols PD, Henson JM, Guckert JB, Nivens DE, White DC: FTIR methods microbial ecology: Analysis of bacteria, bacteria-polymer mixtures and biofilms. J Microbiol Meth 1985, 4:79–94.CrossRef 33. Szalontai B, Nishiyama Y, Gombos Z, Murata N: Membrane dynamics as seen by Fourier transform RAAS inhibitor infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803. The effects of lipid unsaturation and the protein-tolipid ratio. Biochim Biophys Acta 2000, 1509:409–419.PubMedCrossRef 34. Haris PI, Severcan F: FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media. J Mol Catal B Enzym 1999, 7:207–221.CrossRef Competing interests None declared. Authors’ contributions Wang YL and Li B designed the experiments and wrote selleck chemicals the paper. Liu BP, Zhou Q, Wu GX and Ibrahim M performed the experiments. Xie GL, Li HY and Sun GC coordinated the project. All authors

have read and approved the manuscript.”
“Background Cystic fibrosis (CF), an inherited disorder caused by mutations in the gene that encodes the cystic fibrosis Resveratrol transmembrane conductance regulator, affects approximately 30,000 Americans, primarily those of Northern European origin [1, 2]. These mutations cause a deficiency in chloride secretion with ensuing accumulation of thick, stagnant mucus within the lung alveoli of the patients [1–4]. Nutrients in the thick mucus facilitate the colonization of various bacterial pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae[3, 5]. Colonization by these pathogens elicits a strong host inflammatory response which leads to destruction of the lung

tissue and, ultimately, death from respiratory failure [1, 6, 7]. P. aeruginosa is one of the significant pathogens in chronic lung infections of CF patients [1, 8]. Among the different factors that contribute to the virulence of P. aeruginosa is its ability to form a biofilm, a community within which bacteria are attached to a substratum or to each other [9]. Within the biofilm, the bacteria are surrounded by extracellular polymeric substance (EPS), which protects them from the effects of the host immune system and from diverse antibiotics [10–12]. Biofilm development occurs in stages that require specific bacterial factors at each stage. For example, during the initial (attachment) stage of biofilm formation, bacteria depend on both the flagellum-mediated swimming motility and the VX-689 price pili-mediated twitching motility [13]. A number of P.

Most topologies assigned all strains to the same main clades as in the whole genome phylogeny, with a few exceptions: 33-rpoB assigned F. hispaniensis to clade 2 and 19-iglC assigned W. persica to clade 2, in subgroup F. noatunensis subsp. orientalis (in both assignments). This is

an interesting observation as rpoB was recently suggested as an alternative marker to 16S rDNA in metagenomic studies [21]. The level of incompatibility and difference in resolution compared to the whole-genome reference topology were decreased, in some cases by a considerable amount, by selecting an optimal combination of markers. Moreover, topologies based on an optimal set of markers significantly increased the average Selleckchem SCH727965 statistical support (i.e. average bootstrap). Generally, both the degree of compatibility and resolution were improved by concatenating sets of two to seven markers in all possible combinations. However, learn more some combinations, in particular

considering incompatibility, might result in poorer topologies than for an estimated topology based on a single marker. This observation is consistent with previous work where concatenation of sequence data have resulted in S63845 mouse biased phylogenetic estimates [50]. All incompatible phylogenetic signals were removed in topologies based on optimised sets of two to seven markers, in contrast to random concatenation. Totally congruent topologies were obtained by concatenating as few as only two markers (08-fabH and 35-tpiA). These two markers were included in all optimal sets. Hence, by selecting an optimal set of markers, a large improvement in resolution and compatibility can be obtained over random concatenation. An exhaustive search strategy was employed to find the optimal set of markers since the total number of available markers was relatively small. It should be pointed out that the number of possible marker combinations increases rapidly with the number

of markers considered Chloroambucil and soon becomes computationally intractable. As all the 742 gene fragments of the core genome in the analysed population have recently become available in [3], an interesting extension to the current work would be to find the optimal set of markers based on all those genes. Such an optimisation could be carried out by utilising one of the myriad of available optimisation techniques, such as a simulated annealing approach [51, 52]. It should be noted that we do only try to minimize the value of the objective metrics, incongruence or resolution difference, with respect to the whole-genome topology. There is no guarantee that the whole genome topology accurately resembles the true underlying species topology as systematic errors and statistical inconsistencies in the phylogenetic inference method could be amplified when analyzing whole genome data [50, 53–55].

In addition, experiments performed to elucidate the mechanism of APF activity indicate that this frizzled 8-related glycopeptide induces altered expression or phosphorylation of certain proteins that differ in some aspects from those seen in canonical Wnt/frizzled signaling. Downstream signal transducers for Wnt/frizzled signaling include Akt, GSK3β, and β-catenin [39]. The serine threonine kinase Akt, also known as protein kinase B (PKB), is a central regulator of cell proliferation, motility and survival whose activity is often

altered in human malignancies [40]. Akt mediates its downstream effects via phosphorylation/inactivation of GSK3β ser9, with subsequently decreased phosphorylation of the GSK3β target selleck inhibitor β-catenin, AG-881 chemical structure resulting in increased β-catenin nuclear translocation, binding to T-cell factor, and stimulation of gene expression related to cell proliferation and survival [30, 41]. In addition to its association with malignant cell proliferation, increased Akt phosphorylation/activation has also been linked to the invasive properties of bladder cancer cells [40]. The inhibition of Akt ser473 and thr308 phosphorylation

by APF suggests that APF may profoundly inhibit bladder epithelial cell Akt activity, and therefore decrease bladder carcinoma cell invasive potential, as well. GSK3β activity is reduced by phosphorylation of ser9 PTK6 but stimulated by phosphorylation on tyr216 [42], and the downstream effects of Akt activation/phosphorylation during Wnt/frizzled signaling include increased ser9 phosphorylation with decreased activity of GSK3β, decreased GSK3β-inducedβ-catenin ser33,37 phosphorylation, and subsequently decreased β-catenin ubiquitination and

degradation. If as -APF mediated its activity in T24 cells purely by inhibiting canonical Wnt/frizzled signaling (like other secreted frizzled-related cell growth inhibitors), GSK3β ser9 phosphorylation should have been decreased substantially, while tyr216 phosphorylation (which may be mediated by mitogen-activated protein kinase kinase (MEK) 1/2) [43] should not have been affected. Our results, which showed only a very minimal decrease in GSK3β ser9 phosphorylation, but a substantial decrease in GSK3β yr216 phosphorylation, indicate that as -APF: 1) does not mediate its activity purely by regulating Wnt/frizzled canonical signaling; 2) may inhibit GSK3β and additional kinases (such as MEK 1/2); and 3) may mediate its antiproliferative effects in T24 cells via inhibition of Akt, GSK3β, and/or MEK1/2 involving downstream effects on selleck targets in addition to β-catenin.

02 ± 0.64 0.49 ± 0.19 7.5 μM iron chloride (FeCl3) 3.63 ± 0.73 2.49 ± 0.64 15.3 μM hemin 1.72 ± 0.92 0.25 ± 0.18 10 μM potassium PKC412 research buy ferrocyanide ARRY-162 nmr (K4[Fe(CN)6]) (Fe2+) 1.34 ± 1.30 0.38 ± 0.33 10 μM potassium ferricyanide (K3[Fe(CN)6]) (Fe3+) 1.80 ± 2.82 0.93 ± 0.85 10 μM ferric ammonium sulfate (Fe(NH4)(SO4)2) 3.33 ± 2.53 2.02 ± 2.11 50 μM iron citrate (C6H5FeO7) 2.20 ± 0.70 3.47 ± 1.17 300 μM 2,2′-dipyridyl < 0.01 < 0.01 300 μM 2,2'-dipyridyl and 200 μM FeCl3 0.04 ± 0.07 < 0.01 300 μM 2,2'-dipyridyl and 200 μM iron citrate 1.59 ± 1.16 0.04 ± 0.06 a Cells were cultivated in M9 minimal medium including 0.8% (w/v) glucose. Iron sources were added

at the given final concentrations. b The activities were determined for triplicate experiments. Extracts of a hypF mutant, Evofosfamide price which cannot synthesize active

hydrogenases [16], had essentially no hydrogenase enzyme activity and served as a negative control. Extracts of the feoB::Tn5 mutant PM06 grown in M9 medium in the absence of iron had a total hydrogenase activity that was 24% that of the wild type without addition of iron compounds (Table 1). Growth of PM06 in the presence of iron chloride or ferric ammonium sulfate restored hydrogenase activity to levels similar to wild type. The exception was potassium ferricyanide, which failed to restore hydrogenase enzyme activity to wild type levels; instead activity was approximately Methocarbamol 50% of that measured in MC4100 grown without iron supplementation and only 50% of that measured after growth of the wild type with potassium ferricyanide (Table 1). In contrast,

growth of PM06 in the presence of ferrocyanide did not restore hydrogenase activity. Addition of hemin as a source of oxidized iron also failed to restore hydrogenase activity to PM06, presumably because hemin cannot be taken up by E. coli and the oxidized iron is also tightly bound to the porphyrin. Taken together, these results are consistent with the ferrous iron transport system being an important route of iron uptake for hydrogenase biosynthesis in the wild type. Addition of 2, 2′-dipyridyl to the growth medium resulted in total loss of hydrogenase activity of the wild type MC4100 and PM06 (Table 1). Supplementation of 200 μM iron chloride or iron citrate together with 300 μM dipyridyl showed that iron citrate restored 66% of the wild type activity while iron chloride failed to restore activity. None of these additions restored hydrogenase activity to PM06. The activities of Hyd-1 and Hyd-2 can be visualized after non-denaturing PAGE followed by specific activity staining [14]; Hyd-3 is labile and cannot be visualized under these conditions. This method allows a specific analysis of the effect of mutations or medium supplements on Hyd-1 and Hyd-2 activity and it should be noted that this method is only semi-quantitative.

Evidentially, treatment with 1 μM CpG-ODN for 8 h reduced the frequency of FasL-expressing HepG2 cells to 28% and treatment for 24 h decreased the frequency of FasL-expressing HepG2 cells to near 10%. Apparently, treatment with CpG-ODN inhibited the expression of FasL in HepG2 cells in a dose- and time-dependent manner. Figure 1 Treatment with CpG-ODN inhibited the expression of FasL in HepG2 cells in a dose- and time-dependent manner. (A) Dose effect. HepG2 cells were treated with different concentrations of CpG-ODN for 48 h. (B) Time

effect. HepG2 cells were treated with 1 μM CpG-ODN for the indicated time periods. The cells were harvested, and the frequency of FasL-positive cells was determined by FACS analysis. INCB28060 in vitro Data are expressed as mean% ± SEM of each group of the cells from four independent GSK2245840 experiments. *p < 0.05 vs. controls. Effect of CpG-ODN on the Fas expression in Jurkat cells Next,

we tested whether treatment with CpG-ODN could modulate the expression of Fas in Jurkat cells. Jurkat cells were treated with 1 μM CpG-ODN for 24 h. The cells were harvested and the relative levels of Fas mRNA transcripts to control GAPDH were determined by quantitative RT-PCR (Figure 2A). Clearly, the relative levels of Fas mRNA transcripts in the CpG-ODN-treated Jurkat cells were reduced to 65%, as compared with that of unmanipulated controls. Furthermore, CHIR98014 the expression of Fas in Jurkat cells was also examined by flow cytometry analysis. The frequency of Fas-expressing Jurkat cells was significantly reduced from 54% ± 2% to 35% ± 1% (Figure 2B). Therefore, CpG-ODN treatment down-regulated the Fas mRNA transcription and protein expression in Jurkat cells in vitro. Figure 2 Treatment with CpG-ODN inhibited the expression of Fas in Jurkat cells. Jurkat cells

were treated with 1 μM CpG-ODN for 24 h, and the cells were collected. PI-1840 The intracellular expression of Fas was examined by qRT-PCR (A) and FCM (B). Data are expressed as mean% ± SEM of each group of the cells from four separate experiments. *p < 0.05 vs. the controls. Effect of CpG-ODN on the HepG2-mediated Jurkat cell apoptosis Engagement of Fas on the cell membrane by FasL can trigger cell apoptosis. Given that CpG-ODN treatment down-regulated the expression of FasL in HepG2 cells and Fas in Jurkat cells, it is possible that CpG-ODN may modulate the HepG2 cell-mediated Jurkat cell apoptosis. Accordingly, we first treated HepG2 and Jurkat cells with 1 μM CpG-PDN or anti-FasL NOK-2 antibody for 24 h for the preparation of effector and target cells, respectively. Next, we co-cultured the unmanipulated HepG2 and Jurkat cells (positive controls), the NOK-2-treated HepG2 and untreated Jurkat cells, the untreated HepG2 and the NOK-2-treated Jurkat cells, the CpG-ODN-treated HepG2 and untreated Jurkat cells, and the untreated HepG2 and the CpG-ODN-treated Jurkat cells for 24, respectively.

CT is a widespread technique in cryoablated renal tumors monitoring allowing morphologic imaging of the kidney during several enhancement phases, in a tri-phases acquisition. The click here multiphasic acquisition with the new MSCTs provides Wortmannin clinical trial a representation of each component of contrast enhancement (intravascular and extravascular).

Therefore, the use of functional imaging techniques to assess tissue perfusion and permeability allows a more deeply angiogenesis process analysis of the tumor with functional informations that cannot be appreciated from qualitative or quantitative (UH) analysis of static tri-phase contrast enhanced images. Furthermore it implies a margining of factors other than angiogenesis that may influence the quantification of contrast enhancement (e.g. amount of contrast agent, patient weight, cardiac output) [31]. The advent of multislice CT scanner with new perfusion software programs creates a unique opportunity for imaging as a reproducible method to assess, BV-6 in vivo

and more deeply than the qualitative evaluation of contrast enhancement, tumor vascularity for monitoring and possibly predicting clinical response to cryotherapy. Otherwise, the common imaging criteria of lesion shrinkage to assess tumor response to cryotherapy may not be the ideal technique of detecting in vivo activity and clinical outcome of ablation and may be implemented with functional imaging parameters from tumor ablated area to obtain much reliable post-treatment informations. RCC is a highly vascularised tumor with verified correlation between contrast enhancement measures and microvessel density [32] and between its quantification and prognostic information in early-stage of RCC [15, 19]. It is well known that neoangiogenesis is a crucial factor Celecoxib for tumor

cell growth and metastatic potential in cancer disease, inversely related with patient survival [33]. This process is characterized by increased microvessel density and microanatomical changes of new vessels related to fenestration of the basement membrane resulting in anomalous tissue perfusion compared to normal parenchyma and an increase in the permeability to large molecules in blood. Considering that tumor neoangiogenesis induces pathophysiological abnormalities to the hemodynamic environment surrounding the tumor, anomalous tissue perfusion can be qualitatively and quantitative expressed in time-enhancement curves and in colour map measurements in a perfusional contrast-enhanced study [31, 34].

In the hybrid structure of both nanostars and J-aggregates, the pronounced dip at 590 nm (which corresponds to the absorption see more wavelength of the J-aggregates) appears as a result of strong coupling of the excited states of J-aggregates and plasmon modes of the nanostars (Figure 4a, blue curve). The wavelength separation between the two peaks in this spectrum (indicated by arrows in Figure 4) is 61 nm, giving the value of Rabi splitting of 213 meV. This value depends on the total absorbance or, in other words, on the concentration of J-aggregates [27], which, for cyanine dye molecules used in this

work, can be influenced by the addition of charged polyelectrolytes [28]. This is selleck chemicals llc demonstrated in Figure 4a (green curve), where positively charged polyelectrolyte PEI has been added to gold nanostars and to the JC1 molecules. As a result, Rabi splitting energy increased to 260 meV, which is 13% of the total transition

energy (which corresponds to spectral position of the dip), indicating the strong coupling regime between the plasmons www.selleckchem.com/products/epz004777.html and the J-aggregate excitons. To demonstrate the advantage of using Au nanostars for the strong coupling with J-aggregates, it would be instructive to compare the values of the achieved Rabi splitting with that of a hybrid system consisting of J-aggregates and gold nanorods [29] of similar volume as nanostars. Based on the TEM image (Figure 2), the effective volume of nanostars was estimated approximating their inner core part by a sphere to which the spikes are attached. The absorption spectrum of Au nanorods used here (Figure 4b, violet curve) exhibits two main resonances: the red-shifted peak at 766 nm corresponds to the longitudinal surface plasmon resonance, whereas the spectral position of the two other bands spanning over the region between 450 and 650 nm is consistent with the wavelengths of the transverse plasmon modes. The absorption band of J-aggregates of JC1 dye (Figure 4c) falls within the spectral region

of the blue-shifted band of the nanorods. In the hybrid system of Au nanorods and J-aggregates, which was fabricated in a similar fashion as that of the gold nanostars, a dip at 595 nm (Figure 4b, cyan curve) with Rabi splitting of 185 meV is observed, which is a much Endonuclease smaller value than that demonstrated above for the nanostar-based hybrid system. Large number of localized plasmon modes in Au nanostars available for coherent coupling with integrated emitters provides the possibility to observe multiple Rabi splitting for the hybrid system where two (or more) different J-aggregate emitters are strongly coupled to gold nanostars. To demonstrate this possibility, we developed a more complex hybrid system integrating nanostars with J-aggregates of not only JC1 but also S2165 dye, whose absorption band is centered at 637 nm, and thus, more than 30 nm red-shifted with respect to the absorption band of JC1 J-aggregates (Figure 5).

9). 100 mg of the isolated cell

wall material was then added to this solution and incubated over night at 28°C. The sample was then centrifuged and the pellet discarded. After heating (5 min; 100°C), centrifugation (10 min 10,000×g) and dialysis (molecular weight cut off 1000), the sample was freeze-dried. Resuspended lyophilized material was then used for further experiments. Removing LPS from the samples via polymyxin B agarose X. campestris pv. campestris lipopolysaccharides (LPSs) were removed from the elicitor preparation using a batch technique by adding an excess amount of polymyxin B agarose [102] as described in [103]. Upon addition of polymyxin B agarose (Sigma-Aldrich), the samples were shaken and centrifuged. While the pellet

probably containing LPS bound to polymyxin B agarose was discarded, the Caspase Inhibitor VI manufacturer supernatant was used for further analyses. Identification, isolation and characterization of oligosaccharides The selleck chemicals llc analyses of oligosaccharides was performed by HPAEC using a DIONEX GP-40 gradient pump; a Merck-Hitachi D-2000 Chromato Integrator; a DIONEX pulsed amperometric detector and a DIONEX UV detector. Monosaccharide composition of isolated oligosaccharides was analyzed upon acid hydrolysis in trifluoroacetic acid (2 M; 120°C for 2 h). Neutral sugars were separated and identified using an isocratic elution (10 mM sodium hydroxide; flow 1 ml/min) with amperometric detection on a check details CarboPac® PA-100 column. For charged sugars a linear sodium acetate gradient ranging from 0.02 M to 0.5 M under alkaline conditions (0.1 M NaOH) with a flow rate of 1 ml/min was used [75]. Pectate fragments were separated using a sodium acetate gradient (ranging from 0.01 M to 1.0 M with a plateau of 10 min. at a concentration 0.7 M sodium acetate; 0.1

M NaOH; CarboPac® PA-100 column; flow 1 ml/min). For the identification of pectate fragments Racecadotril a pectate standard was generated by digestion of pectin (Pectin esterified, Sigma P-9561) by pectate lyase (Sigma P-7052). The isolation of pectate fragments was carried out under the conditions described above, but a semi-preparative column (CarboPac® PA-1; flow 2.5 ml/min) was used. MALDI-TOF MS of isolated oligosaccharides Crude extracts were analyzed on a Bruker ultraflex I MALDI-TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) in the negative–ion mode. Samples were analyzed in the linear and in the reflector TOF. Gentisic acid was used as matrix. For sample preparation, 1 μl saturated gentisic acid solution was mixed with 1 μl of 50 mg ml–1 crude extract lyophilisate dissolved in demineralized water. One microliter of this mixture was dropped onto the MALDI target. Determination of the oxidative burst reaction in plant cell suspension cultures The detection of the oxidative burst was performed using the H2O2-dependent chemiluminescence reaction described by Warm [104].