About -rV ending verbs in the Sakhalin dialect of Ainu

Table S2. Abbreviations of peroxidase gene names used for the peroxidase-catalase superfamily. (XLSX 54 kb

Exploring Site-Specific N‑Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure–function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered <i>Nicotiana benthamiana</i> plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from <i>N. benthamiana</i> displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs

Investigation of Ion Binding in Chlorite Dismutases by Means of Molecular Dynamics Simulations

Chlorite dismutases are prokaryotic heme <i>b</i> oxidoreductases that convert chlorite to chloride and dioxygen. It has been postulated that during turnover hypochlorite is formed transiently, which might be responsible for the observed irreversible inactivation of these iron proteins. The only charged distal residue in the heme cavity is a conserved and mobile arginine, but its role in catalysis and inactivation is not fully understood. In the present study, the pentameric chlorite dismutase (Cld) from the bacterium <i>Candidatus Nitrospira defluvii</i> was probed for binding of the low spin ligand cyanide, the substrate chlorite, and the intermediate hypochlorite. Simulations were performed with the enzyme in the ferrous, ferric, and compound I state. Additionally, the variant R173A was studied. We report the parametrization for the GROMOS force field of the anions ClO^–, ClO₂^–, ClO₃^–, and ClO₄^– and describe spontaneous binding, unbinding, and rebinding events of chlorite and hypochlorite, as well as the dynamics of the conformations of Arg173 during simulations. The findings suggest that (i) chlorite binding to ferric NdCld occurs spontaneously and (ii) that Arg173 is important for recognition and to impair hypochlorite leakage from the reaction sphere. The simulation data is discussed in comparison with experimental data on catalysis and inhibition of chlorite dismutase

Clinical score of the paws of DA rats.

<p>While all data correspond to one experiment the experimental groups are displayed in three diagrams for reasons of comprehensibility. PIA was induced on d0 in all female DA rats except the healthy control group (empty squares; n = 5). The application of pristane led to an acute joint swelling followed by a short intermediate recovery and the subsequent development of chronic arthritic symptoms. For each treatment group the mean score of the rats for each scoring day is given with the SEM (Kruskal-Wallis test followed by Dunn’s Post Hoc test). <b>(A):</b> While the positive control (filled squares; n = 14) received saline, the treatment control received 0.05 mg MTX/kg via injection on five consecutive days. Thereby the treatment started either on d1 (light grey empty triangles; n = 15) or individually when the animals reached a score of five (light grey filled triangles; n = 14). The early treatment with MTX i.p. (treatment control) inhibited the joint swelling and redness significantly both in the acute and in the chronic phase while a late application of this treatment had no effect. <b>(B):</b> EGCG (0.1% w/v) was continuously given via drinking water again starting on d1 (grey empty circles; n = 10) or on the day the animal reached a score of five (grey filled circles; n = 10). The early treatment with EGCG p.o. considerably inhibited the joint swelling and redness in the acute and in the chronic phase just while a late application of EGCG had no effect. <b>(C):</b> 10 mg EGCG/kg were injected on five consecutive days starting either on d1 (grey empty triangle; n = 15) or on the day the animal reached a score of five (grey filled triangle; n = 15). The i.p. application of EGCG had no beneficial effect on the course of the disease and, upon late application, even led to a significantly stronger joint swelling and redness in the chronic phase.</p

Histological analysis of metacarpal sections on d155 after pristane injection.

<p>The displayed pictures are representative examples for tissue sections obtained from healthy animals (A), from the saline-treated control group (B) and from EGCG treated groups (C: EGCG p.o. early and D: EGCG i.p. late). For experimental details regarding arthritis-induction and treatment see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152518#pone.0152518.g001" target="_blank">Fig 1</a>. In all cases the metacarpus section of the forepaw was stained with Nuclear Fast Red, Aniline blue and Orange G. (A): In the healthy control group no signs for joint swelling and destruction were observed. Different tissue types (bone cartilage: blue, muscle: red, skin: red-orange) were clearly distinguishable. (B): In contrast, in the positive control a massive joint swelling was observable. Moreover, especially the bone and cartilage tissue was considerably destructed (dark blue regions). (C): Only mild cartilage destruction was apparent in the metacarpi of rats early and orally treated with EGCG whereas rats from the EGCG i.p. late treatment group (D) mostly showed patho-histological signs of arthritis very similar to the saline-treated positive control (B). The scale bars correspond to 1 mm (overviews) and 250 μm (inserts).</p

EGC-derived formation of Compound II and regeneration of native MPO.

<p>Experimental conditions are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152518#pone.0152518.g006" target="_blank">Fig 6</a>. <b>(A):</b> As illustrated for 20 μM EGC time traces recorded at 456 nm are suitable to follow both transitions. By using fitting functions <i>k</i>_obs values were determined and re-plotted against the flavonoid concentration. <b>(B):</b> For the formation of Compound II a second-order rate constant of 5.02 x 10⁶ M^-1 s^-1 was determined <b>(C):</b> for the subsequent regeneration of native MPO a rate of 1.04 x 10³ M^-1 s^-1 was determined. The time traces <b>(A)</b> used for the determination of <i>k</i>_obs values represent averaged curves from three to four independent measurements. The linear dependence between the <i>k</i>_obs values and the EGC concentration is illustrated by the R² values obtained during the application of linear fitting functions <b>(B)</b> and <b>(C)</b>.</p

Chlorinating MPO activity in the time course of pristane-induced arthritis.

<p>For each animal the geometric mean of the APF-dependent fluorescence intensity distribution was determined as a sign for the HOCl-producing MPO activity of the blood neutrophils. <b>(A):</b> While in the healthy control group (n = 5) relatively stable values for the MPO activity were found throughout the experiment in the saline-treated positive control (n = 14), a strong increase in the HOCl production especially in the acute and in the chronic disease phase was detected. In the group with early MTX treatment (n = 15) the therapeutic effect of the drug was nicely reflected by considerably lower MPO activity values. <b>(B):</b> As the application of EGCG only reduced the arthritic symptoms upon early oral administration (n = 10), in this group also a strong reduction of the MPO activity values was observed. In the other EGCG treatment groups the HOCl production recurrently exceeded the range of normal MPO activity determined on d0 (dashed lines). Yet the differences were never statistically significant.</p

Plasma concentrations of α1AGP on d21 after pristane injection.

<p>The acute-phase protein was quantified in blood samples taken on d21 from animals of the healthy negative control (no pristane injection, n = 5), the saline-treated positive control (n = 14) and the rats treated with EGCG p.o. starting on d1 (n = 10). For experimental details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152518#pone.0152518.g001" target="_blank">Fig 1</a>. From the blood samples plasma was obtained and stored at -80°C till analysis via ELISA. Significant differences were determined by applying a one-way ANOVA followed by a Holm-Sidak test. Thereby (***) corresponds to p values ≤ 0.001 and (**) corresponds to p ≤ 0.01. While in the healthy control only about 150 μg/ml of the protein were found, in the positive control the amount of α1AGP was about 4.5 times higher. Yet in the EGCG-treated group the protein levels were only elevated 3.5-fold.</p

Spectral transitions of MPO Compound I by EGC.

<p>All measurements were performed at 22°C in 100 mM phosphate buffer, pH 7.0. Final concentrations of MPO and H₂O₂ were 2 μM and 25 μM, respectively. Representative spectra from at least three independent experiments are shown. The indicated time points were selected from 50 to 100 recorded spectra. Compound I was pre-formed by incubating the enzyme with a 12.5-fold H₂O₂ excess for 50 ms. <b>(A):</b> In the absence of EGC a slow H₂O₂-derived transition to compound II was observed within the 5 s. <b>(B):</b> However, in the presence of 20 μM EGC (final concentration) this Compound II formation was considerably faster and followed by a second transition where native MPO is regenerated from Compound II.</p

Chronification of the PIA in DA rats.

<p>The data were taken from the animal experiment displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152518#pone.0152518.g001" target="_blank">Fig 1</a>. For each animal the lowest score during the remission phase (d40–d50) was determined. If this value subsequently increased by eight or more the development of a chronic arthritic phase in the animal was assumed. The relative amount of animals which became chronic within the different pristane-treated experimental groups is shown in black. Only the early i.p. application of MTX or the early p.o. administration of EGCG led to a significantly lower amount of chronic arthritic animals. Significant differences between the experimental groups were tested by applying the Kruskal-Wallis test. Thereby (*) corresponds to p values ≤ 0.05.</p