In situ localization of micropollutants and associated stress response in Populus nigra leaves.

Micropollutants and emerging organic contaminants (EOCs) have been widely studied in terms of persistance, removal, human risk assessment, toxicology, etc. Mass spectrometry imaging (MSI) offers the possibility of following the fate of a single pesticide in a plant leaf or a drug in the whole body of an animal, organ by organ. However, the admissibility of chronic low doses of complex mixtures for the ecosystem has not been assessed. How do micropollutants diffuse in the environment? How do living organisms cope with chronic exposure to a low dose of diverse micropollutants? Is there a cocktail effect or a chance for hormesis? Combining mass spectrometry imaging (MSI) and targeted and nontargeted liquid chromatography coupled to mass spectrometry (LC-MS), we attempt to answer these questions. We investigate the diversity of micropollutants at the exit of a water treatment facility, their diffusion in sludge and black poplar (Populus nigra), and their impact on a living organism. We reveal a specific tissue localization of micropollutants in peripheral leaf tissues, and an associated stress response from the plant, with stress hormones and tissue degradation markers induced in the plant growing near the water efflux.

Endothelium-independent vasorelaxant effect of a Berberis orthobotrys root extract via inhibition of phosphodiesterases in the porcine coronary artery.

BACKGROUND: Berberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension. HYPOTHESIS: This study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism. STUDY DESIGN: An aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases. RESULTS: The aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP- and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, CaCl2, and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMP-hydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with IC50 values ranging from 40 to 130µg/ml. CONCLUSION: The butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP- and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

Lymphocytic esophagitis in children.

BACKGROUND: Lymphocytic esophagitis (LE) is a term recently suggested for the finding of >20 intraepithelial lymphocytes/high-power field in an esophageal biopsy with no more than a rare granulocyte. Two prior studies of LE suggested an association of LE with Crohn's disease (CD) in young patients, but there has been no systematic review of a large pediatric cohort to determine the prevalence and clinical associations of LE in children. METHODS: All esophageal biopsies performed at a tertiary care pediatric medical center in 2005 were identified (580 biopsies from 545 unique patients). A blinded histologic review was performed to identify LE cases (>50 intraepithelial lymphocytes/high-power field; <1 granulocyte/50 intraepithelial lymphocytes). Clinical characteristics, endoscopic findings, and follow-up data for each case were reviewed independently by a pediatric gastroenterologist. RESULTS: Thirty-one patients with LE (5.7%) and 49 patients with CD (8.9%) were found among the 545 patients. Six of the 31 LE patients (19%) and 43 of the 514 non-LE patients (8.4%) had CD (P < 0.05). The remaining LE patients had various other clinical diagnoses with no significant clinical correlates. LE was identified in 6 of 49 patients with CD (12.2%) and 25 of 496 patients without CD (5.0%) (P < 0.05). Patients with both LE and CD had a more prominent lymphocytic infiltrate than LE patients without CD. CONCLUSIONS: LE seems to be more prevalent in children than in adults and has a significant association with CD in this age group.

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions.

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25 degrees C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l(-1), respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties.

Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by 1H and 19F nuclear magnetic resonance spectroscopy.

After polymerization filamentous actin (F-actin) still shows a number of rather narrow 1H NMR signals in its Mg2+ form which are quenched when Mg2+ is replaced by Ca2+. These resonances originate from mobile residues in F-actin. For assignment of these resonances three different strategies were used, the fluorine labeling of Cys-374 by 4-(perfluoro-tert-butyl)phenyliodoacetamide, binding studies with antibodies (Fab) against the seven N-terminal amino acids of actin, and two-dimensional 1H NMR spectroscopy of a highly concentrated F-actin sample. In contrast to the effects detected earlier by 1H NMR spectroscopy, 19F NMR spectroscopy of actin labeled at its C-terminal cysteine shows no significant spectral changes in dependence on the divalent ion present. In its G- (globular) form a strong, narrow 19F resonance can be observed at 15.06 ppm (relative to the external standard trifluoroacetic acid) which is broadened substantially after polymerization of actin. At 283 K the corresponding transverse relaxation time T2 decreases from 16.7 ms to approximately 0.6 ms. These data suggest that the highly mobile residues observed by 1H NMR spectroscopy do not originate from the C-terminus. Binding of Fab directed against the N-terminal amino acids of actin to Mg-F-actin leads to the disappearing of the 1H NMR resonances assigned to a mobile domain in F-actin. This indicates that the mobile region probably comprises the N-terminal amino acids. By homonuclear two-dimensional 1H NMR spectroscopy it was finally possible to sequentially assign the resonances of the mobile domain of F-actin. It turned out that amino acids 1-22 are in a highly mobile state in Mg-F-actin. The nuclear Overhauser effect data indicate that, rather surprisingly, in this high mobility state some of the beta-pleated structure is still conserved. The population of F-actin protomers in the M- (mobile) state can be obtained from the NMR spectra and was determined under different experimental conditions. In the presence of 150 mM KCl approximately half of the protomers in Mg-F-actin are in the M-state. This number is largely independent of the pH in the range studied (pH 7.2-7.8) and of the temperature in range studied (283-310 K). The equilibrium constant KMI for the equilibrium between the I- and M-states is approximately 1.3 under these conditions.

The ternary complex of DNase I, actin and thymosin beta4.

We have recently described a method for identifying contact sites between actin and thymosin beta4 (Tbeta4) by following spectrophotometrically the extent and kinetics of distinct, thiol-specific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et a]. (1996) J. Biol. Chem. 271, 1301-1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin-DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the KD value of Tbeta4 from the actin-DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tbeta4. In conclusion, DNase I and Tbeta4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin beta4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tbeta4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tbeta4 and actin by X-ray analysis.

Cross-link between cys 374 and cys 10 of actin abolishes polymerizability and allows study of the properties of the "F-actin monomer".

Actin cross-linked between cys 374 and cys 10 via a disulfide-containing bridge, c-A, is completely unpolymerizable even in the presence of phalloidin. Upon the addition of dithiothreitol, c-A polymerizes with high yield, indicating that denaturation due to the modification was almost absent. In the present study we show that cross-linked actin is a useful model for studying the properties of monomeric actin under polymerization conditions. Addition of salt, for example, produced fluorescence changes possibly reflecting conformational transitions but did not lead to the development of phalloidin binding capacity. Cross-linking of the two cysteine residues also caused a decrease in the nucleotide exchange rate by a factor of ca. 3, an effect that was fully reversed by the addition of KCl. Cross-linked actin inhibits DNase I to the same extent as G-actin and binds thymosin beta 4 and profilin as shown by cross-linking studies. Capping capacity for the barbed end of the filament was not observed, although it might have been expected from the fact that both ends of the cross-link are anchored to subdomain 1. Using the 61-FITC derivative of c-A we showed that c-A is able to bind to myosin S1 with a KD in the microM range. In agreement with this, c-A shows actomyosin ATPase activity with a Kapp comparable to that of F-actin, but a Vmax decreased by a factor of ca. 11. The c-A myosin S1 complex provides the hitherto smallest model of actomyosin, which appears promising for crystallization and X-ray analysis.

Identification of contact sites in the actin-thymosin beta 4 complex by distance-dependent thiol cross-linking.

Binding sites of actin and thymosin beta 4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys10, Cys374, or the sulfur atom of the ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S), the actin derivatives were reacted with synthetic thymosin beta 4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys374 of actin and Cys6 of thymosin beta 4, indicating that the N terminus of thymosin beta 4 is in close proximity (< or = 9.2 A) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin beta 4 analogs when the cross-linkers were anchored at Cys10 of actin. A second contact site was identified by cross-linking of Cys17 and Cys28 in thymosin beta 4 with the ATP gamma S derivative bound to actin, indicating that the hexamotif of thymosin beta 4 (positions 17-22) is in close proximity (< or = 9.2 A) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin beta 4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.

Complete 1H nuclear magnetic resonance assignments and structural characterization of a fusion protein of the alpha-amylase inhibitor tendamistat with the activation domain of the human immunodeficiency virus type 1 Tat protein.

Complete sequence-specific assignments of the 1H-NMR spectrum of a fusion protein of the alpha-amylase inhibitor tendamistat from Streptomyces tendae and the activation domain of Tat from human immunodeficiency virus type 1 (HIV-1) was obtained by homonuclear two-dimensional NMR methods. The protein behaves as expected for an ideal fusion protein: the flexible linker allows an almost completely decoupled motion of the subunits of the protein and the two subunits show almost no mutual interaction. In the tendamistat part, small structural distortions due to exchange of the carboxy-terminal leucine propagate mainly via the hydrogen bonds of the beta-sheet and the disulfide bond. The Tat part of the protein contains the seven cysteine residues of full-length Tat. The fusion protein was expressed in Streptomyces lividans and exported. During the export to the extracellular space disulfide bonds are created by the expressing cells, only one sulfhydryl group remains accessible for sulfhydryl reagents. Although a unique, dominant conformation with a specific disulfide bonding pattern exists, a significant conformational variation can be observed including cis-proline peptide bonds, which may indicate smaller populations with alternative disulfide bonding patterns.