11-Dehydro-thromboxane B2, a stable thromboxane metabolite, is a full agonist of chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) in human eosinophils and basophils

Thromboxane (TX) A(2), a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB(2), which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB(2), but not the TXA(2) analogue U46,619 or TXB(2), activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB(2) was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D(2) has been shown to be its principal ligand. 11-Dehydro-TXB(2) induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD(2) but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB(2) were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB(2)- and PGD(2)-induced shape change. 11-Dehydro-TXB(2) also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB(2) had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB(2) is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB(2)/CRTH2axis may be of therapeutic relevance.     

Modeling mechanical stresses as a factor in the etiology of benign vocal fold lesions

Vocal fold tissue lesions such as nodules and polyps are thought to develop in response to mechanical stress that occurs during vocal fold collision. Two computational models of vocal fold collision during voice production are used to investigate this hypothesis. A one-dimensional lumped mass model, whose parameters are derived from vocal fold tissue dimensions and material properties, predicts stress perpendicular to the direction of impact (normal stress). A previously published three-dimensional finite element model that incorporates the same dimensions and properties predicts the entire stress tensor. The hypothesis is supported by predictions from the finite element model that three components of normal stress and one component of shear stress are increased during collision in the typical location of lesions (i.e. the center of the superior medial edge of the vocal fold in the middle of the vibrating and contact region). The lumped mass model predicts that mechanical stress is negatively correlated with mucosal thickness (increased by voice warm-up and hydration), is positively correlated with driving force (proportional to voice intensity), and is affected by voice production method. These relationships are consistent with clinical observations of vocal fold lesion risk factors and have implications for improving prevention and treatment of benign vocal fold lesions.     

Single- and dual-parameter FRET kinase probes based on pleckstrin

Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create diverse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d; microscopy and image processing take 2 h.     

The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth

Studies have suggested that diets rich in polyphenols such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D1 levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract.     

The SuUR gene influences the distribution of heterochromatic proteins HP1 and SU(VAR)3–9 on nurse cell polytene chromosomes of Drosophila melanogaster

We have investigated the distribution of three heterochromatic proteins [SUppressor of UnderReplication (SUUR), heterochromatin protein 1 (HP1), and SU(VAR)3–9] in chromosomes of nurse cells (NCs) and have compared the data obtained with the distribution of the same proteins in salivary gland (SG) chromosomes. In NC chromosomes, the SU(VAR)3–9 protein was found in pericentric heterochromatin and at 223 sites on euchromatic arms, while in SG chromosomes, it was mainly restricted to the chromocenter. In NC chromosomes, the HP1 and SUUR proteins bind to 331 and 256 sites, respectively, which are almost twice the number of sites in SG chromosomes. The distribution of the HP1 and SU(VAR)3–9 proteins depends on the SuUR gene. A mutation in this gene results in a dramatic decrease in the amount of SU(VAR)3–9 binding sites in autosomes. In the X chromosome, these sites are relocated in comparison to the SuUR ⁺, and their total number only varies slightly. HP1 binding sites are redistributed in chromosomes of SuUR mutants, and their overall number did not change as considerably as SU(VAR)3–9. These data together point to an interaction of these three proteins in Drosophila NC chromosomes.Electronic Supplementary Material Supplementary material is available for this article at.     

Crystal structure of NirD,the small subunit of the nitrite reductase NirbD from Mycobacterium tuberculosis at 2.0 Å resolution

NirD is part of the nitrite reductase complex NirBD that catalyses the reduction of nitrite to NH₃ in nitrate assimilation and anaerobic respiration. The crystal structure analysis of NirD from Mycobacterium tuberculosis shows a double β‐sandwich fold. NirD is related in three‐dimensional structure and sequence to the Rieske proteins; however, it does not contain any Fe–S cluster or other cofactors that might be involved in electron transfer. A cysteine residue at the protein surface, conserved in NirD homologues lacking the iron–sulfur cluster might be important for the interaction with NirB and possibly stabilize one of the Fe–S centers in this subunit. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Identification of a redox-sensitive switch within the JAK2 catalytic domain

Four cysteine residues (Cys866, Cys917, Cys1094, and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(NΔ661)rJAK2), which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated by in vitro autokinase assays and by in situ autophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pretreatment with dithiothreitol or o-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.     

Renal IL-17 expression in human ANCA-associated glomerulonephritis

Interleukin-17A (IL-17) promotes inflammatory renal tissue damage in mouse models of crescentic glomerulonephritis, including murine experimental autoimmune anti-myeloperoxidase glomerulonephritis, which most likely depends on IL-17-producing Th17 cells. In human anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, however, the cellular sources of IL-17 remain to be elucidated. Therefore, we analyzed human kidney biopsies of active necrotizing and crescentic ANCA-associated glomerulonephritis by immunohistochemistry using an IL-17-specific antibody and by immunofluorescent colocalization with cell type markers. We detected numerous IL-17-expressing (IL-17(+)) cells in the glomeruli and in the tubulointerstitium. Unexpectedly, most of these IL-17(+) cells were polymorphonuclear neutrophilic granulocytes, while IL-17(+) T cells and IL-17(+) mast cells were present at significantly lower frequencies. IL-17 was not detected in other infiltrating or resident kidney cells. In those patients who had not received immunosuppressive treatment before biopsy, serum creatinine levels were positively correlated with tubulointerstitial IL-17(+) neutrophils as well as IL-17(+) T cells. Furthermore, we could demonstrate that purified human blood neutrophils expressed IL-17 protein and released it upon stimulation in vitro. In conclusion, these results support a pathogenic role for IL-17 in human ANCA-associated glomerulonephritis. Our data suggest that in the acute stage of the disease neutrophils may act as an important immediate-early innate source of IL-17 and may thereby initiate and promote ongoing renal inflammation. IL-17 may thus be a target for treating acute ANCA-associated glomerulonephritis.     

Mitochondrial Dysfunction—A Pharmacological Target in Alzheimer's Disease

Increasing evidences suggest that mitochondrial dysfunction plays an important role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Alterations of mitochondrial efficiency and function are mainly related to alterations in mitochondrial content, amount of respiratory enzymes, or changes in enzyme activities leading to oxidative stress, mitochondrial permeability transition pore opening, and enhanced apoptosis. More recently, structural changes of the network are related to bioenergetic function, and its consequences are a matter of intensive research. Several mitochondria-targeting compounds with potential efficacy in AD including dimebon, methylene blue, piracetam, simvastatin, Ginkgo biloba, curcumin, and omega-3 polyunsaturated fatty acids have been identified. The majority of preclinical data indicate beneficial effects, whereas most controlled clinical trials did not meet the expectations. Since mitochondrial dysfunction represents an early event in disease progression, one reason for the disappointing clinical results could be that pharmacological interventions might came too late. Thus, more studies are needed that focus on therapeutic strategies starting before severe disease progress.     

Brain Isoprenoids Farnesyl Pyrophosphate and Geranylgeranyl Pyrophosphate are Increased in Aged Mice

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23?months) as compared to middle-aged mice (12?months) and young mice (3?months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.