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961.
962.
Anatoly F. Vanin Alexander P. Poltorakov Vasak D. Mikoyan Lioudmila N. Kubrina Ernst van Faassen 《Nitric oxide》2006,15(4):295-311
The in vivo mechanism of NO trapping by iron-dithiocarbamate complexes is considered. Contrary to common belief, we find that in biological systems the NO radicals are predominantly trapped by ferric iron-dithiocarbamates. Therefore, the trapping leads to ferric mononitrosyl complexes which are diamagnetic and cannot be directly detected with Electron Paramagnetic Resonance spectroscopy. The ferric mononitrosyl complexes are far easily reduced to ferrous state with L-cysteine, glutathione, ascorbate or dithiocarbamate ligands than their non-nitrosyl counterpart. When trapping NO in oxygenated biological systems, the majority of trapped nitric oxide is found in diamagnetic ferric mononitrosyl iron complexes. Only a minority fraction of NO is trapped in the form of paramagnetic ferrous mononitrosyl iron complexes with dithiocarbamate ligands. Subsequent ex vivo reduction of biological samples sharply increases the total yield of the paramagnetic mononitrosyl iron complexes. Reduction also eliminates the overlapping EPR spectrum from Cu(2+)-dithiocarbamate complexes. This facilitates the quantification of yields from NO trapping. 相似文献
963.
Exudates from the brown algaeCaepidium antarcticum andDesmarestia sp. were investigated for their ability to associate with hydrophobic pollutants such as polychlorinated biphenyls (PCB s).
The percentage of PCB associated with algal exudates ranged from 79% for decachlorobiphenyl to 23% for the pentachlorobiphenyl
congener No. 95. Exudates from the tested brown algae may therefore alter the bioavailability of PCBs in natural or artificial
ecosystems. 相似文献
964.
965.
Holger Rupp Jörg Rinklebe Sebastian Bolze Ralph Meissner 《Ecological Engineering》2010,36(10):1439-1447
The Elbe River, Germany, has received heavy metals and arsenic from the discharge of urban industrial, and agricultural effluent. During periods of inundation, these contaminants were transported with water into floodplain ecosystems, where they settled and accumulated predominantly in depressions and low-lying terraces. Markedly elevated arsenic concentration in soil solution during floods exceeded the inspection value of 10 μg L?1 of the German soil protection ordinance. Highly variable hydrological conditions in floodplains can affect the dynamics of pollutants. The study of processes controlling the dynamics of pollutants is challenging because the results are required to answer both scientific and practical questions regarding protection of groundwater and plants, sustainable management of floodplains or explain the fate of environmentally harmful substances.Our experiments in small groundwater lysimeter and biogeochemical microcosms tended to yield similar results regarding the functional relationships among the investigated site parameters. But the results of the field experiments, carried out at a floodplain site of the middle course of the Elbe River, Germany, are often characterized by complex and varying factors. Whereas arsenic tended to be mobilized during flooding due to decreasing redox potential (EH), chromium showed the opposite trend, with peak concentrations at the highest EH values. Our approach at three different spatiotemporally scale levels, ranging from 23 days (microcosms) to two-and-a-half years (field soil hydrological facility) allows us to overcome process interferences observed in field studies. 相似文献
966.
José Carlos Quintela Francisco García-del Portillo Ernst Pittenauer Günter Allmaier Miguel A. de Pedro 《Journal of bacteriology》1999,181(1):334-337
Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)2 bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear 252californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H+ and Na+ ions and in the negative ion mode with H− and CN− ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. (Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. (Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)2 bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A204 of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.
Open in a separate windowaDR5 and DR10 to DR13 were analyzed in both the positive and negative ion modes. Muropeptides DR1 to DR4 and DR6 to DR9 were analyzed in the positive mode only due to the small amounts of sample available. bMass difference between measured and calculated quasimolecular ion values. c[(Measured mass−calculated mass)/calculated mass] × 100. dN-Acetylglucosamine. eN-Acetylmuramitol. f(1→6)Anhydro-N-acetylmuramic acid. Structural assignments of muropeptides DR1, DR2, DR8 to DR10, and DR12 deserve special comments. The low m/z value measured for DR1 (700.1) fitted very well with the value calculated for GlucNAc–MurNAc–l-Ala–d-Glu (699.69). Even smaller was the mass deduced for DR9 from the m/z value of the molecular ion of the sodium adduct (702.1) (Fig. (Fig.2).2). The mass difference between DR1 and DR9 (19.9 mass units) was very close indeed to the calculated difference between N-acetylmuramitol and the (1→6)anhydro form of MurNAc (20.04 mass units). Therefore, DR9 was identified as GlucNAc–(1→6)anhydro-MurNAc–l-Ala–d-Glu (Fig. (Fig.1).1). Muropeptides with (1→6)anhydro muramic acid have been identified in mureins from diverse origins (10, 15, 17, 19), indicating that it might be a common feature among peptidoglycan-containing microorganisms. Open in a separate windowFIG. 2Positive-ion linear PDMS of muropeptide DR9. Muropeptide DR9 was purified, desalted by HPLC, and subjected to PDMS to determine the molecular mass. The masses for the dominant molecular ions are indicated.The measured m/z value for the [M+Na]+ ion of DR8 was 1,521.6, very close to the mass calculated for a cross-linked dimer without one disaccharide moiety (1,520.53) (Fig. (Fig.1;1; Table Table1).1). Such muropeptides, also identified in T. thermophilus HB8 and other bacteria (18, 19), are most likely generated by the enzymatic clevage of MurNAc–l-Ala amide bonds in murein by an N-acetylmuramyl–l-alanine amidase (21). In particular, DR8 could derive from DR11. The difference between measured m/z values for DR8 and DR11 was 478.7, which fits with the mass contribution of a disaccharide moiety (480.5) within the mass accuracy of the instrument.The m/z values for muropeptides DR2, DR10, and DR12 supported the argument for structures in which the two d-Ala residues from the d-Ala–d-Ala C-terminal dipeptide were lost, leaving Orn as the C-terminal amino acid.The position of one Gly residue in muropeptides DR2, DR8, and DR10 to DR13 could not be formally demonstrated. One of the Gly residues could be at either the N- or the C-terminal positions. However, the N-terminal position seems more likely. The structure of the basic muropeptide (DR7), with a (Gly)2 acylating the δ-NH2 group of Orn, suggests that major muropeptides should present a (Gly)2 dipeptide. The scarcity of DR3 and DR6, which unambiguously have Gly as the C-terminal amino acid (Fig. (Fig.1),1), supports our assumption.Molar proportions for each muropeptide were calculated as proposed by Glauner et al. (10) and are shown in Table Table1.1. For calculations the structures of DR10 to DR13 were assumed to be those shown in Fig. Fig.1.1. The degree of cross-linkage calculated was 47.2%. Trimeric muropeptides were rather abundant (8 mol%) and made a substantial contribution to total cross-linkage. However, higher-order oligomers were not detected, in contrast with other gram-positive bacteria, such as Staphylococcus aureus, which is rich in such oligomers (8). The proportion of muropeptides with (1→6)anhydro-muramic acid (5 mol%) corresponded to a mean glycan strand length of 20 disaccharide units, which is in the range of values published for other bacteria (10, 17).The results of our study indicate that mureins from D. radiodurans and T. thermophilus HB8 (19) are certainly related in their basic structures but have distinct muropeptide compositions. In accordance with the phylogenetic proximity of Thermus and Deinococcus (11), both mureins are built up from the same basic monomeric subunit (DR7 in Fig. Fig.1),1), are cross-linked by (Gly)2 bridges, and have (1→6)anhydro-muramic acid at the termini of glycan strands. Most interestingly, Deinococcus and Thermus are the only microorganisms identified at present with the murein chemotype A3β as defined by Schleifer and Kandler (20). Nevertheless, the differences in muropeptide composition were substantial. Murein from D. radiodurans was poor in d-Ala–d-Ala- and d-Ala–Gly-terminated muropeptides (2.2 and 2.4 mol%, respectively) but abundant in Orn-terminated muropeptides (23.8 mol%) and in muropeptides with a peptide chain reduced to the dipeptide l-Ala–d-Glu (18 mol%). In contrast, neither Orn- nor Glu-terminated muropeptides have been detected in T. thermophilus HB8 murein, which is highly enriched in muropeptides with d-Ala–d-Ala and d-Ala–Gly (19). Furthermore, no traces of phenyl acetate-containing muropeptides, a landmark for T. thermophilus HB8 murein (19), were found in D. radiodurans. Cross-linkage was definitely higher in D. radiodurans than in T. thermophilus HB8 (47.4 and 27%, respectively), largely due to the higher proportion of trimers in the former.The similarity in murein basic structure suggests that the difference between D. radiodurans and T. thermophilus HB8 with respect to the Gram reaction may simply be a consequence of the difference in the thickness of cell walls (2, 3, 23). Interestingly, D. radiodurans murein turned out to be relatively simple for a gram-positive organism, possibly reflecting the primitive nature of this genus as deduced from phylogenetic trees (11). Our results illustrate the phylogenetic proximity between Deinococcus and Thermus at the cell wall level but also point out the structural divergences originated by the evolutionary history of each genus. 相似文献
TABLE 1
Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radioduransMuropeptidea | Ion | m/z
| Δmb | Error (%)c | Muropeptide composition
| Muropeptide abundance (mol%) | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Calculated | Measured | NAGd | NAMe | Glu | Orn | Ala | Gly | |||||
DR1 | [M+H]+ | 699.69 | 700.1 | 0.41 | 0.06 | 1 | 1 | 1 | 0 | 1 | 0 | 12.0 |
DR2 | [M+H]+ | 927.94 | 928.3 | 0.36 | 0.04 | 1 | 1 | 1 | 1 | 1 | 2 | 5.7 |
DR3 | [M+Na]+ | 1,006.97 | 1,007.5 | 0.53 | 0.05 | 1 | 1 | 1 | 1 | 1 | 3 | 3.0 |
DR4 | [M+Na]+ | 963.95 | 964.6 | 0.65 | 0.07 | 1 | 1 | 1 | 1 | 2 | 1 | 2.5 |
DR5 | [M+H]+ | 999.02 | 999.8 | 0.78 | 0.08 | 1 | 1 | 1 | 1 | 2 | 2 | 27.7 |
[M−H]− | 997.00 | 997.3 | 0.30 | 0.03 | ||||||||
DR6 | [M+Na]+ | 1,078.5 | 1,078.8 | 0.75 | 0.07 | 1 | 1 | 1 | 1 | 2 | 3 | 2.4 |
DR7 | [M+H]+ | 1,070.09 | 1,071.0 | 0.90 | 0.08 | 1 | 1 | 1 | 1 | 3 | 2 | 2.2 |
DR8 | [M+Na]+ | 1,520.53 | 1,521.6 | 1.08 | 0.07 | 1 | 1 | 2 | 2 | 4 | 4 | 2.2 |
DR9 | [M+Na]+ | 701.64 | 702.1 | 0.46 | 0.03 | 1 | 1f | 1 | 0 | 1 | 0 | 5.0 |
DR10 | [M+H]+ | 1,907.94 | 1,907.8 | 0.14 | 0.01 | 2 | 2 | 2 | 2 | 3 | 4 | 10.1 |
[M−H]− | 1,905.92 | 1,906.6 | 0.68 | 0.04 | ||||||||
DR11 | [M+H]+ | 1,979.01 | 1,979.1 | 0.09 | 0.01 | 2 | 2 | 2 | 2 | 4 | 4 | 19.1 |
[M−H]− | 1,977.00 | 1,977.3 | 0.30 | 0.02 | ||||||||
DR12 | [M+H]+ | 2,887.93 | 2,886.5 | −1.43 | −0.05 | 3 | 3 | 3 | 3 | 5 | 6 | 4.4 |
[M−H]− | 2,885.91 | 2,885.8 | −0.11 | −0.01 | ||||||||
DR13 | [M+H]+ | 2,959.00 | 2,957.8 | −1.20 | −0.04 | 3 | 3 | 3 | 3 | 6 | 6 | 3.6 |
[M−H]− | 2,956.99 | 2,955.9 | −1.09 | −0.04 |
967.
968.
Sunasara KM Cramer SM Hauer CR Rupp RG Shoup VA 《Archives of biochemistry and biophysics》1999,372(2):248-260
The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the question of the in vitro activity of these variants. Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF variants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively. Peak 4, which is chromatographically a single peak, contained three variants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak 5 had norleucine at position 31 instead of methionine. All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants. 相似文献
969.
Peptide-based fluoromethyl ketones have been considered for many years to be highly specific caspase inhibitors distinctly blocking the progress of apoptosis in a variety of systems. Here we demonstrate that these compounds can significantly reduce rhinovirus multiplication in cell culture. In their methylated forms they block eIF4GI cleavage in vivo and in vitro and inhibit the activity of picornaviral 2A proteinases. 相似文献
970.
Swietnicki W O'Brien S Holman K Cherry S Brueggemann E Tropea JE Hines HB Waugh DS Ulrich RG 《The Journal of biological chemistry》2004,279(37):38693-38700
Binary complexes formed by components of the Yersinia pestis type III secretion system were investigated by surface plasmon resonance (SPR) and matrix-assisted laser desorption time-of-flight mass spectrometry. Pairwise interactions between 15 recombinant Yersinia outer proteins (Yops), regulators, and chaperones were first identified by SPR. Mass spectrometry confirmed over 80% of the protein-protein interactions suggested by SPR, and new binding partners were further characterized. The Yop secretion protein (Ysc) M2 of Yersinia enterocolitica and LcrQ of Y. pestis, formerly described as ligands only for the specific Yop chaperone (Syc) H, formed stable complexes with SycE. Additional previously unreported complexes of YscE with the translocation regulator protein TyeA and the thermal regulator protein YmoA and multiple potential protein contacts by YscE, YopK, YopH, and LcrH were also identified. Because only stably folded proteins were examined, the interactions we identified are likely to occur either before or after transfer through the injectosome to mammalian host cells and may have relevance to understanding disease processes initiated by the plague bacterium. 相似文献