Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ∼50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30–50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.     

Inactivation of C3a by a monocarboxypeptidase present in culture supernatants of stimulated guinea pig peritoneal macrophages

Hog C3a, as well as its derivative C3a-desArg were not found to act cytotoxically on starch gel-induced guinea pig peritoneal macrophages. Likewise, neither peptide significantly modified the secretion of N-acetyl-beta-D-glucosaminidase from these cells. However, C3a rapidly lost its spasmogenic activity during incubation in serum-free macrophage cultures and less rapidly in cellfree supernatants collected from cultured macrophages. The following results indicate that C3a is converted into its spasmogenically inactive derivative C3a-desArg by a macrophage-derived monocarboxypeptidase. The inactivated C3a product does not differ from native C3a in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; it elutes from CM cellulose in the same position as purified C3a-desArg; and it is devoid of the carboxyl-terminal arginyl residue of C3a, but still contains the carboxyl-terminal sequence of C3a-desArg as determined by analysis after treatment with carboxypeptidases B or Y. Furthermore, inactivation of C3a in supernatants of macrophage cultures is completely blocked by the specific carboxypeptidase inhibitors guanidinopropylsuccinic acid and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid in final concentrations of 10 mM and 2.1 mM, respectively. The monocarboxypeptidase is apparently supplied by biosynthesis of new material but is not stored as a preformed enzyme because cycloheximide markedly inhibits its expression.     

ESR-Untersuchungen an röntgenbestrahlten Leucin-Salzen

Zusammenfassung Die Radikalausbeute in röntgenbestrahltem L-Leucin -HCl beträgt bei ZimmertemperaturG _R=2,2. Kinetische Messungen ergaben Hinweise, daß bei Zimmertemperatur mehr als ein Radikaltyp vorliegt. Aus der Dosiseffektkurve sowie der Kinetik und thermischen Stabilität der Radikale nach höheren Bestrahlungsdosen wird geschlossen, daß ihre Stabilisierung in der Festkörpermatrix durch eine mit steigender Dosis zunehmende Anhäufung von Rekombinationsprodukten verringert wird.

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ESR-studies on X-irradiated salts of leucineII. Radical yield and radical kinetics in irradiated L-Leucine · HCl

Summary The free radical yield in L-Leucine · HCl following X-irradiation at room temperature isG _R=2,2. The kinetic study shows more than one radical type. From the shape of the dose-yield-curve as well as the kinetics and the annealing behaviour of the radicals after higher irradation doses we assume, that the stabilisation of the radicals by the surrounding lattice is reduced with increasing accumulation of recombination products.

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1. Mitteilung: ESR-Untersuchungen zur Radikalstruktur in bestrahltem L-Leucin-HCl. Biophysik5, 14 (1968).

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Herrn Prof. Dr. habil.Kh. Lohs möchten wir für die Unterstützung und Diskussion dieser Arbeit danken. Frl. G.Klein danken wit für die umsichtige und sorgfältige technische Mitarbeit.     

Human glomerular mesangial cells inactivate leukotriene B4 by reduction into dihydro-leukotriene B4 metabolites

Due to its potent chemotactic properties leukotriene B4 is an important mediator of inflammatory reactions. Cultured human kidney mesangial cells converted exogenously added leukotriene B4 efficiently into three different more lipophilic metabolites, two of them probably representing dihydro-leukotriene B4 isomers. This represents an alternative metabolic pathway, in contrast to leukotriene B4 omega-oxidation found in human polymorphonuclear leukocytes. Both dihydro-leukotriene B4 isomers had nearly completely lost their ability to induce leukocyte chemotaxis as compared to leukotriene B4.     

Morphological parameters affecting false lumen thrombosis following type B aortic dissection: a systematic study based on simulations of idealized models

Type B aortic dissection (TBAD) carries a high risk of complications, particularly with a partially thrombosed or patent false lumen (FL). Therefore, uncovering the risk factors leading to FL thrombosis is crucial to identify high-risk patients. Although studies have shown that morphological parameters of the dissected aorta are related to FL thrombosis, often conflicting results have been reported. We show that recent models of thrombus evolution in combination with sensitivity analysis methods can provide valuable insights into how combinations of morphological parameters affect the prospect of FL thrombosis. Based on clinical data, an idealized geometry of a TBAD is generated and parameterized. After implementing the thrombus model in computational fluid dynamics simulations, a global sensitivity analysis for selected morphological parameters is performed. We then introduce dimensionless morphological parameters to scale the results to individual patients. The sensitivity analysis demonstrates that the most sensitive parameters influencing FL thrombosis are the FL diameter and the size and location of intimal tears. A higher risk of partial thrombosis is observed when the FL diameter is larger than the true lumen diameter. Reducing the ratio of the distal to proximal tear size increases the risk of FL patency. In summary, these parameters play a dominant role in classifying morphologies into patent, partially thrombosed, and fully thrombosed FL. In this study, we point out the predictive role of morphological parameters for FL thrombosis in TBAD and show that the results are in good agreement with available clinical studies.

     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

General relation between variance-time curve and power spectral density for point processes exhibiting 1/f β-fluctuations,with special reference to heart rate variability

Counting statistics in the form of the variance-time curve provides an alternative to spectral analysis for point processes exhibiting 1/f ^β-fluctuations, such as the heart beat. However, this is true only for β<1. Here, the case of general β is considered. To that end, the mathematical relation between the variance-time curve and power spectral density in the presence of 1/f ^β-noise is worked out in detail. A modified version of the variance-time curve is presented, which allows us to deal also with the case β?1. Some applications to the analysis of heart rate variability are given.     

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.