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.     

Infectious diarrhea of infant rats produced by a rotavirus-like agent.

During the investigation of an outbreak of diarrhea in suckling rats, a virus morphologically identical to but antigenically distinct from rotaviruses was identified. The disease was characterized clinically by erythema and cracking and bleeding of the perianal skin associated with the excretion of poorly formed fecal pellets, liquid, and gas. Light microscopy-observable changes consisted of small intestinal villous atrophy, villous epithelial necrosis, and villous epithelial syncytial cell formation. The cytoplasm of the epithelial syncytial cells contained large numbers of 80-nm viral particles that were often associated with reticular aggregates of electron-dense material. Viral infection principally involved the luminal one-fourth to one-third of the intestinal villi as determined by indirect immunofluorescence. This rotavirus-like agent contained 11 double-stranded RNA segments; however, the migration pattern of these segments in polyacrylamide gels differed from the electrophoretic pattern which is characteristic of the typical rotaviruses. The agent had a buoyant density in CsCl of 1.36 to 1.4 g/cm3 and was labile at pH 3 and at 56 degrees C; however, infectivity of viral inocula was not altered by extensive treatment with ether or by pH 5 buffers. This disease, which we have named infectious diarrhea of infant rats, is the first recognized viral diarrhea of rats and appears to be a good model for the study of the recently recognized group of atypical rotaviruses.     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Determination of methotrexate in human urine at nanomolar levels by high-performance liquid chromatography with column switching

An high-performance liquid chromatographic method with column switching for the detection of less than 4 ng of methotrexate in the urine of oncologic nurses is described. Urine samples were purified by solid-phase extraction on silica-bonded phenyl columns, eluting impurities with ethyl acetate. After elution from the column, the analyte was concentrated ten-fold, evaporating the solvent. On a strong anion-exchange column (Nucleosil 100 SB), methotrexate was separated from the remaining interfering substances, was then switched to a reversed-phase column (LiChrospher 100 RP-18e), and finally eluted by a linear gradient in a solvent system consisting of ammonium formate buffer (pH 2.7) and acetonitrile. Absorbance was monitored at 310 nm. This method has proved to be suitable for detecting traces of methotrexate in urine in order to individualize risks and to reduce further the occupational safety hazard for hospital personnel.     

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.     

The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.     

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.