A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model.

Nitric oxide (NO), an important cell signaling molecule, is considered a marker of inflammatory response and is elevated in asthmatics. This study investigated the effects of montelukast (a leukotriene receptor antagonist) on iNOS expression and activity in a Brown Norway (BN) rat allergic inflammation model and in L2 lung epithelial cells. Allergic inflammation was induced by ovalbumin (OVA) injection in BN rats followed by treatment with either montelukast or dexamethasone (DX). Allergen inhalation was performed, and post-allergen Penh was measured 5 min after the challenge. Cysteinyl leukotriene levels were measured in bronchoalveolar lavage (BAL) fluid and lung iNOS expression and activity determined. These parameters were also measured in cytokine stimulated L2 lung epithelial cells. iNOS expression was significantly higher in OVA challenged rats compared to the naive, DX, and montelukast treated groups, as confirmed by immunohistochemistry and Western blot analysis. However, no significant differences in NOS activity were found. Cysteinyl leukotriene measured in BAL was significantly higher in all OVA challenged rats compared to naive controls. Incubation of L2 cells with a mixture of interferon gamma (IFNgamma), lipopolysaccharide (LPS), and tumor necrosis factor (TNFalpha) resulted in high levels of nitrite formation resulting from iNOS induction. Treatment of cytokine stimulated cells with DX or montelukast significantly decreased iNOS expression and activity. No detectable cysteinyl leukotrienes were found in the supernatant fluid of L2 cells. This study confirms the ability of montelukast to modulate iNOS function and raises the possibility that changes in iNOS expression and activity may occur via pathways independent of cysteinyl leukotrienes.     

Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the “two factor theory.” The present study deals with a third, largely ignored component—mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70–110, 250–500, and 1,000–1,250 µm, as a means of altering the surface area with which the cells come in contact. Post‐thaw evaluation included motility at 0 h and after 3 h at 37°C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra‐container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area‐to‐volume ratio, the intra‐container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing. Biotechnol. Bioeng. 2009; 104: 719–728 © 2009 Wiley Periodicals, Inc.     

Trace quantification of 1-octacosanol and 1-triacontanol and their main metabolites in plasma by liquid–liquid extraction coupled with gas chromatography–mass spectrometry

A method for the simultaneous determination of 1-octacosanol and 1-triacontanol and their main metabolites in rat plasma was developed. The procedure involved ethanolic NaOH saponification of the sample, acidification, liquid–liquid extraction, and derivatization of the analytes to its trimethylsilylether/ester, followed analysis by gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring (SIM) mode. Quantification was performed by the internal standard method using betulin. The method had a good linearity over the range 8.4–540 ng/ml (r ≥ 0.998) and showed an excellent intra-day (R.S.D. = 0.59–3.06%) and inter-day (R.S.D. = 2.99–5.22%) precision according to the acceptance criteria. The detection limits ranged between 1.32 and 3.47 ng/ml. The method was applied successfully to study the total plasmatic concentration of 1-octacosanol, octacosanoic acid, 1-triacontanol, and triacontanoic acid, after an oral dose of policosanols mixture, using plasma samples of 100 μl.     

Time-Warp–Invariant Neuronal Processing

Fluctuations in the temporal durations of sensory signals constitute a major source of variability within natural stimulus ensembles. The neuronal mechanisms through which sensory systems can stabilize perception against such fluctuations are largely unknown. An intriguing instantiation of such robustness occurs in human speech perception, which relies critically on temporal acoustic cues that are embedded in signals with highly variable duration. Across different instances of natural speech, auditory cues can undergo temporal warping that ranges from 2-fold compression to 2-fold dilation without significant perceptual impairment. Here, we report that time-warp–invariant neuronal processing can be subserved by the shunting action of synaptic conductances that automatically rescales the effective integration time of postsynaptic neurons. We propose a novel spike-based learning rule for synaptic conductances that adjusts the degree of synaptic shunting to the temporal processing requirements of a given task. Applying this general biophysical mechanism to the example of speech processing, we propose a neuronal network model for time-warp–invariant word discrimination and demonstrate its excellent performance on a standard benchmark speech-recognition task. Our results demonstrate the important functional role of synaptic conductances in spike-based neuronal information processing and learning. The biophysics of temporal integration at neuronal membranes can endow sensory pathways with powerful time-warp–invariant computational capabilities.     

The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

Background

The topoisomerases Top1, Top2α and Top2β are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2β are subject to proteasomal degradation, this phenomena was not demonstrated for Top2α.

Methodology/Principal Findings

We show here that Top2α is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2β. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2α degradation, increases the persistence of Top2α-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2α in-vitro and cellular overexpression of Bmi1 increases drug induced Top2α ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2α ubiquitination and drug-induced Top2α degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.

Conclusions/Significance

The discovery that poisoned Top2α is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.     

Structural interactions between FXYD proteins and Na+,K+-ATPase: alpha/beta/FXYD subunit stoichiometry and cross-linking

Interactions of rat FXYD4 (corticosteroid hormone-induced factor (CHIF)), FXYD2 (gamma), or FXYD1 (phospholemman (PLM)) proteins with rat alpha1 subunits of Na(+),K(+)-ATPase have been analyzed by co-immunoprecipitation and covalent cross-linking. In detergent-solubilized membranes from HeLa cells expressing both gamma and CHIF or CHIF and hemagglutinin A-tagged CHIF, mixed complexes of CHIF and gamma or CHIF and hemagglutinin A-tagged CHIF with alpha/beta subunits are undetectable. This implies that the alpha/beta/FXYD protomer is the major species in detergent solution. A lipid-soluble cysteine-cysteine bifunctional reagent, dibromobimane, cross-links CHIF to alpha in colonic membranes but not gamma or PLM to alpha in kidney or heart membranes, respectively. Sequence comparisons of the FXYD proteins suggested that Cys-49 in the trans-membrane segment of CHIF could be involved. In detergent-solubilized HeLa cell membranes, dibromobimane cross-links wild-type CHIF to alpha but not the C49F mutant, and also the corresponding F36C mutant but not wild-type gammab, and F48C but not wild-type PLM. C140S, C338A, C804A, and C966S mutants of the alpha subunit have been expressed. Only the C140S mutant prevents cross-linking with CHIF. The data demonstrated the proximity of trans-membrane segments of CHIF, gamma, and PLM to M2 of alpha. Molecular modeling is consistent with location of the trans-membrane segment of all FXYD proteins between M2, M6, and M9 and the proximity of Cys-49 of CHIF or Phe-36 of gamma with Cys-140 of M2. Cross-linking also demonstrated CHIF-alpha and CHIF-beta proximities in extra-membrane regions, similar to the evidence for gamma-alpha and gamma-beta cross-links.     

Small‐scale spatial variability in the distribution of ectomycorrhizal fungi affects plant performance and fungal diversity

The effects of spatial heterogeneity in negative biological interactions on individual performance and species diversity have been studied extensively. However, little is known about the respective effects involving positive biological interactions, including the symbiosis between plants and ectomycorrhizal (EM) fungi. Using a greenhouse bioassay, we explored how spatial heterogeneity of natural soil inoculum influences the performance of pine seedlings and composition of their root‐associated EM fungi. When the inoculum was homogenously distributed, a single EM fungal taxon dominated the roots of most pine seedlings, reducing the diversity of EM fungi at the treatment level, while substantially improving pine seedling performance. In contrast, clumped inoculum allowed the proliferation of several different EM fungi, increasing the overall EM fungal diversity. The most dominant EM fungal taxon detected in the homogeneous treatment was also a highly beneficial mutualist, implying that the trade‐off between competitive ability and mutualistic capacity does not always exist.