Azethoxyl nitroxide spin labels. ESR studies involving thiourea crystals, model membrane systems and chromatophores, and chemical reduction with ascorbate and dithiothreitol

Trans- and cis-azethoxyl nitroxides , , and can be trapped in the cavities of thiourea crystals. The presence of a single gauche conformation on either side of the pyrrolidine ring within the crystals was indicated by the ESR spectra. Rotation about the long molecular axis then corresponds approximately to y-axis motion of the nitroxide moiety. Proxyl nitroxides in which the nitroxide group is located on the penultimate carbon of long chain lipids can also be trapped and were shown to adopt the azethoxyl conformation in the thiourea crystals.The measured ΔA values (A_| − A) of oriented egg lecithin multilayers containing trans- and cis-azethoxyl nitroxides and were quite small, consistent with the unique orientation of the nitroxide principal axes with respect to the long axis of the molecule. The ΔA values for a series of lipids bearing a label near the terminus of the chain were very similar to that of , showing that the azethoxyl conformation is likely the predominant one in these labels in orienting systems.Computer simulations of the ESR spectra of and in egg lecithin vesicles provided values for molecular orientation and motion parameters consistent with those expected from a consideration of molecular models in the extended (all trans) conformation.Azethoxyl nitroxides have also proven useful in the investigation of motion restricted (boundary) lipid in a lipid-protein system. Thus, the values (69 ± 10%) for the amount of boundary lipid in the chromatophore membranes from Rhodopseudomonas sphaeroides as determined using trans- and cis- are in good agreement with values using 16-doxylstearic acid (64 ± 3%). The fact that all three labels show about the same fraction of boundary lipid in this system indicates that the lipid binding sites are relatively insensitive to the geometry of the lipid chain. Also, both and appear to be able to detect a third lipid environment not seen with the doxyl fatty acid. The apparent fluidity of this component lies between that of boundary and bilayer lipid. The unique orientation of the nitroxide principal axes with respect to the long molecular axis in azethoxyl nitroxides and allows detection of hindrance to rotation about the long molecular axis, in contrast to the analogous doxyl and proxyl fatty acids.Comparative reduction studies using ascorbate and dithiothreitol indicate that azethoxyl nitroxides are slightly more resistant toward reduction than proxyl nitroxides and much more resistant than doxyl nitroxides.     

Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.     

Use of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation

We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.     

Critical role for T cells in Sephadex-induced airway inflammation: pharmacological and immunological characterization and molecular biomarker identification

Intratracheal instillation of Sephadex particles is a convenient model for assessing the impact of potential anti-inflammatory compounds on lung eosinophilia thought to be a key feature in asthma pathophysiology. However, the underlying cellular and molecular mechanisms involved are poorly understood. We have studied the time course of Sephadex-induced lung eosinophilia, changes in pulmonary T cell numbers, and gene and protein expression as well as the immunological and pharmacological modulation of these inflammatory indices in the Sprague Dawley rat. Sephadex increased T cell numbers (including CD4(+) T cells) and evoked a pulmonary eosinophilia that was associated with an increase in gene/protein expression of the Th2-type cytokines IL-4, IL-5, and IL-13 and eotaxin in lung tissue. Sephadex instillation also induced airway hyperreactivity to acetylcholine and bradykinin. A neutralizing Ab (R73) against the alphabeta-TCR caused 54% depletion of total (CD2(+)) pulmonary T cells accompanied by a significant inhibition of IL-4, IL-13 and eotaxin gene expression together with suppression (65% inhibition) of eosinophils in lung tissue 24 h after Sephadex treatment. Sephadex-induced eosinophilia and Th2 cytokine gene and/or protein expression were sensitive to cyclosporin A and budesonide, compounds that inhibit T cell function, suggesting a pivotal role for T cells in orchestrating Sephadex-induced inflammation in this model.     

Dissociation by steroids of eosinophilic inflammation from airway hyperresponsiveness in murine airways

Background

The link between eosinophils and the development of airway hyperresponsiveness (AHR) in asthma is still controversial. This question was assessed in a murine model of asthma in which we performed a dose ranging study to establish whether the dose of steroid needed to inhibit the eosinophil infiltration correlated with that needed to block AHR.

Methods

The sensitised BALB/c mice were dosed with vehicle or dexamethasone (0.01–3 mg/kg) 2 hours before and 6 hours after each challenge (once daily for 6 days) and 2 hours before AHR determination by whole-body plethysmography. At 30 minutes after the AHR to aerosolised methacholine the mice were lavaged and differential white cell counts were determined. Challenging with antigen caused a significant increase in eosinophils in the bronchoalveolar lavage (BAL) fluid and lung tissue, and increased AHR.

Results

Dexamethasone reduced BAL and lung tissue eosinophilia (ED₅₀ values of 0.06 and 0.08 mg/kg, respectively), whereas a higher dose was needed to block AHR (ED₅₀ of 0.32 mg/kg at 3 mg/ml methacholine. Dissociation was observed between the dose of steroid needed to affect AHR in comparison with eosinophilia and suggests that AHR is not a direct consequence of eosinophilia.

Conclusion

This novel pharmacological approach has revealed a clear dissociation between eosinophilia and AHR by using steroids that are the mainstay of asthma therapy. These data suggest that eosinophilia is not associated with AHR and questions the rationale that many pharmaceutical companies are adopting in developing low-molecular-mass compounds that target eosinophil activation/recruitment for the treatment of asthma.     

Invertebrate community response to fire and rodent activity in the Mojave and Great Basin Deserts

Recent increases in the frequency and size of desert wildfires bring into question the impacts of fire on desert invertebrate communities. Furthermore, consumer communities can strongly impact invertebrates through predation and top‐down effects on plant community assembly. We experimentally applied burn and rodent exclusion treatments in a full factorial design at sites in both the Mojave and Great Basin deserts to examine the impact that fire and rodent consumers have on invertebrate communities. Pitfall traps were used to survey invertebrates from April through September 2016 to determine changes in abundance, richness, and diversity of invertebrate communities in response to fire and rodent treatments. Generally speaking, rodent exclusion had very little effect on invertebrate abundance or ant abundance, richness or diversity. The one exception was ant abundance, which was higher in rodent access plots than in rodent exclusion plots in June 2016, but only at the Great Basin site. Fire had little effect on the abundances of invertebrate groups at either desert site, with the exception of a negative effect on flying‐forager abundance at our Great Basin site. However, fire reduced ant species richness and Shannon's diversity at both desert sites. Fire did appear to indirectly affect ant community composition by altering plant community composition. Structural equation models suggest that fire increased invasive plant cover, which negatively impacted ant species richness and Shannon's diversity, a pattern that was consistent at both desert sites. These results suggest that invertebrate communities demonstrate some resilience to fire and invasions but increasing fire and spread of invasive due to invasive grass fire cycles may put increasing pressure on the stability of invertebrate communities.     

Does evaluation of the ligamentous compartment enhance diagnostic utility of sacroiliac joint MRI in axial spondyloarthritis?

IntroductionInflammation of the sacroiliac joints (SIJ) is a fundamental clinical feature of axial spondyloarthritis (SpA). The anatomy of the irregularly shaped SIJ is complex with an antero-inferior cartilaginous compartment containing central hyaline and peripheral fibrocartilage, and a dorso-superior ligamentous compartment. Several scoring modules to systematically assess SIJ magnetic resonance imaging (MRI) in SpA have been developed. Nearly all of them are based on the cartilaginous joint compartment alone. However, there are only limited data about the frequency of inflammatory lesions in the ligamentous compartment and their potential diagnostic utility in axial SpA. We therefore aimed to evaluate the ligamentous compartment on sacroiliac joint MRI for lesion distribution and potential incremental value towards diagnosis of SpA over and above the traditional assessment of the cartilaginous compartment alone.MethodsTwo independent cohorts of 69 and 88 consecutive back pain patients ≤50 years were referred for suspected SpA (cohort A) or acute anterior uveitis plus back pain (cohort B). Patients were classified according to rheumatologist expert opinion based on clinical, radiographic and laboratory examination as having nonradiographic axial SpA (nr-axSpA; n = 51), ankylosing spondylitis (n = 34), or nonspecific back pain (NSBP; n = 72). Five blinded readers assessed SIJ MRI globally for presence/absence of SpA. Bone marrow edema (BME) and fat metaplasia were recorded in the cartilaginous and ligamentous compartment. The incremental value of evaluating the ligamentous additionally to the cartilaginous compartment alone for diagnosis of SpA was graded qualitatively. We determined the lesion distribution between the two compartments, and the impact of the ligamentous compartment evaluation on diagnostic utility.ResultsMRI bone marrow lesions solely in the ligamentous compartment in the absence of lesions in the cartilaginous compartment were reported in just 0–2.0/0–4.0 % (BME/fat metaplasia) of all subjects. Additional assessment of the ligamentous compartment was regarded as essential for diagnosis in 0 and 0.6 %, and as contributory in 28.0 and 7.7 % of nr-axSpA patients in cohorts A and B, respectively. Concomitant BME in both compartments was evident in 11.6–42.0 % of nr-axSpA and 2.1–2.4 % of NSBP patients.ConclusionAssessing the ligamentous compartment on SIJ MRI provided no incremental value for diagnosis of axial SpA. However, concomitant BME in both compartments may help discriminate nr-axSpA from NSBP.