Sphingosine-1-Phosphate Receptor Type 4 (S1P4) Is Differentially Regulated in Peritoneal B1 B Cells upon TLR4 Stimulation and Facilitates the Egress of Peritoneal B1a B Cells and Subsequent Accumulation of Splenic IRA B Cells under Inflammatory Conditions

Background: Gram-negative infections of the peritoneal cavity result in profound modifications of peritoneal B cell populations and induce the migration of peritoneal B cells to distant secondary lymphoid organs. However, mechanisms controlling the egress of peritoneal B cells from the peritoneal cavity and their subsequent trafficking remain incompletely understood. Sphingosine-1-phosphate (S1P)-mediated signaling controls migratory processes in numerous immune cells. The present work investigates the role of S1P-mediated signaling in peritoneal B cell trafficking under inflammatory conditions. Methods: Differential S1P receptor expression after peritoneal B cell activation was assessed semi‑quantitatively using RT-PCR in vitro. The functional implications of differential S1P₁ and S1P₄ expression were assessed by transwell migration in vitro, by adoptive peritoneal B cell transfer in a model of sterile lipopolysaccharide (LPS)‑induced peritonitis and in the polymicrobial colon ascendens stent peritonitis (CASP) model. Results: The two sphingosine-1-phosphate receptors (S1PRs) expressed in peritoneal B cell subsets S1P₁ and S1P₄ are differentially regulated upon stimulation with the TLR4 agonist LPS, but not upon PMA/ionomycin or B cell receptor (BCR) crosslinking. S1P₄ deficiency affects both the trafficking of activated peritoneal B cells to secondary lymphoid organs and the positioning of these cells within the functional compartments of the targeted organ. S1P₄ deficiency in LPS-activated peritoneal B cells results in significantly reduced numbers of splenic innate response activator B cells. Conclusions: The S1P-S1PR system is implicated in the trafficking of LPS-activated peritoneal B cells. Given the protective role of peritoneal B1a B cells in peritoneal sepsis, further experiments to investigate the impact of S1P₄-mediated signaling on the severity and mortality of peritoneal sepsis are warranted.     

Linear ion trap for second-order Doppler shift reduction in frequency standard applications

The authors have designed and are presently testing a novel linear ion trap that permits storage of a large number of ions with reduced susceptibility to the second-order Doppler effect caused by the RF confining fields. This new trap should store about 20 times the number of ions as a conventional RF trap with no corresponding increase in second-order Doppler shift from the confining field. In addition, the sensitivity of this shift to trapping parameters, i.e., RF voltage, RF frequency, and trap size, is greatly reduced. The authors have succeeded in trapping mercury ions and xenon ions in the presence of helium buffer gas. Trap times as long as 2x 10(3) s have been measured.     

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

Evidence is presented that temperature-sensitive Saccharomyces cerevisiae mutants, impaired in dolichol kinase (Sec59p) or dolichyl phosphate mannose synthase (Dpm1p) activity have an aberrant cell wall composition and ultrastructure. The mutants were oversensitive to Calcofluor white, an agent interacting with the cell wall chitin. In accordance with this, chemical analysis of the cell wall alkali-insoluble fraction indicated an increased amount of chitin and changes in the quantity of beta1,6- and beta1,3-glucan in sec59-1 and dpm1-6 mutants. In order to unravel the link between the formation of dolichyl phosphate and dolichyl phosphate mannose and the cell wall assembly, we screened a yeast genomic library for a multicopy suppressors of the thermosensitive phenotype. The RER2 and SRT1 genes, encoding cis-prenyltransferases, were isolated. In addition, the ROT1 gene, encoding protein involved in beta1,6-glucan synthesis (Machi et al., 2004) and protein folding (Takeuchi et al., 2006) acted as a multicopy suppressor of the temperature-sensitive phenotype of the sec59-1 mutant. The cell wall of the mutants and of mutants bearing the multicopy suppressors was analysed for carbohydrate and mannoprotein content. We also examined the glycosylation status of the plasma membrane protein Gas1p, a beta1,3-glucan elongase, and the degree of phosphorylation of the Mpk1/Slt2 protein, involved in the cell wall integrity pathway.     

Cr-doped Zr, Si-mesoporous molecular sieves as catalysts of CH2Cl2 oxidation

Zirconium-containing mesoporous silica of nominal Si/Zr ratio in the range 40–5 have been synthesized using dodecylamine as a structure-directing surfactant. The samples were characterized with PXRD, SEM/EDX, BET, CEC measurement and chemical analysis. The materials were used as supports for chromium species incorporated by means of cation exchange procedure. Gradual substitution of Si by Zr in the mesoporous framework results in an increasing structural disorder, increasing cation exchange capacity and increasing capacity for incorporation of Cr. All Cr-doped ZrMMSx catalysts are active in the deep oxidation of methylene chloride reaching 100% conversion at temperature ≥400°C. The catalytic performance depends strongly on the catalyst composition. The role of particular components in determining the catalytic activity and selectivities to various products is discussed.     

Effect of steroids and nitric oxide on pituitary hormone release in ovariectomized, peripubertal rats

The purpose of this study was to determine the effects of the duration of steroid depletion on the steroid-induced luteinizing hormone and prolactin surges in ovariectomized, peripubertal female rats. Additionally, the role of nitric oxide (NO) in mediating the surge responses was determined. Peripubertal, 6-week-old, female Sprague-Dawley rats were ovariectomized. One or three weeks later, animals were injected with 17 beta-estradiol (50 microg, sc) followed 48 h later by progesterone (2.5 mg, sc). Effects of NO were examined by administering L-arginine (300 mg/kg, ip). The response of ovariectomized, adult females to steroid treatment was also determined.One and three weeks after ovariectomy, steroid replacement produced an LH and prolactin surge in peripubertal animals. However, both the magnitude and duration of the LH surge was greater 3 weeks after ovariectomy. While L-arginine significantly enhanced the magnitude of the LH surge 1 week after ovariectomy, by 3 weeks L-arginine caused a decrease in the duration, but not the magnitude of the surge. In contrast, L-arginine did not affect either the magnitude or duration of the prolactin surge one week after ovariectomy, but diminished the magnitude after 3 weeks of steroid depletion. In adults, steroids induced significant increases in both LH and prolactin. These results demonstrate that sensitivity to NO stimulation of LH, but not prolactin secretion, is modulated by the duration of gonadal steroid hormone depletion. The differences in the responsiveness of LH and prolactin to steroid-induced stimulation in peripubertal animals demonstrate that these hormones are regulated by NO through different mechanisms.     

An Experimental Toolbox for Structure-Based Hit Discovery for P. aeruginosa FabF,a Promising Target for Antibiotics

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.     

Preliminary study on the application of visible–near infrared spectroscopy and chemometrics to classify Riesling wines from different countries

Visible (VIS) and near infrared (NIR) spectroscopy combined with chemometrics was used in an attempt to classify commercial Riesling wines from different countries (Australia, New Zealand, France and Germany). Commercial Riesling wines (n = 50) were scanned in the VIS and NIR regions (400–2500 nm) in a monochromator instrument, in transmission mode. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and stepwise linear discriminant analysis (SLDA) based on PCA scores were used to classify Riesling wines according to their country of origin. Full cross validation (leave-one-out) was used as the validation method when classification models were developed. PLS-DA models correctly classified 97.5%, 80% and 70.5% of the Australian, New Zealand and European (France and Germany) Riesling wines, respectively. SLDA calibration models correctly classified 86%, 67%, 67% and 87.5% of the Australian, New Zealand, French and German Riesling wines, respectively. These results demonstrated that the VIS and NIR spectra contain information that when used with chemometrics allow discrimination between wines from different countries. To further validate the ability of VIS–NIR to classify white wine samples, a larger sample set will be required.     

The Role of pH in the Mechanism of .OH Radical Induced Oxidation of Nicotine

The ^.OH radical induced oxidation of nicotine was studied using pulse radiolysis techniques from pH 1 to 13.6. Theoretical calculations were used to help interpret the experimental results. The bond dissociation enthalpies of all of the C H bonds of nicotine were determined using DFT calculations, coupled with the isodesmic reaction. From time-dependent density functional response theory, estimates were obtained of the location of the dominant transient absorption bands (λ_max), their intensities (electronic oscillator strength, f), and the electronic composition of these transitions. OH radicals as well as other potent oxidants reacted with free nicotine through separated, concerted electron proton transfer, leading mostly to the formation of an alpha-aminoalkyl radical located on the C_2′ carbon of the aliphatic ring ( A_2′ ). Protonated nicotine underwent hydrogen atom abstraction at the C_2′ and N_1′ positions, resulting in the formation of the conjugate acid of A_2′ ( A_2′H⁺ ) and the alkylamine radical cation ( N⁺ ), respectively. Doubly protonated nicotine underwent the same reaction pathways, leading to two corresponding conjugate acid species, protonated at the aromatic nitrogen position: PyrH⁺A_2′H⁺ and PyrH⁺N⁺ _. All these radicals interconverted between each other through hydrolytic reactions. The radical A_2′ and its conjugate acid PyrH⁺A_2′ absorbed 10 times stronger than the N⁺ species, based on calculations of f. From the growth of the transient absorption of A_2′ (λ_max=330 nm, ε=8080 M ⁻¹ cm⁻¹), second-order rate constants were determined: k_(OH+Nic)=6.7×10⁹ M ⁻¹ s ⁻¹, k_(OH+NicH)=1.0×10⁹ M ⁻¹ s⁻¹. The alpha-aminoalkyl radicals decayed by disproportionation to form iminium cations 1 – 5 , which contributed to an increase in the specific conductivity of the basic solutions of nicotine following electron pulse irradiation.     

Optimisation of the anti-Trypanosoma brucei activity of the opioid agonist U50488

Screening of the Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)-(1R,2R)-U50488, a weak opioid agonist with an EC(50) value of 59 nM as determined in our T. brucei in vitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure-activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.