Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.     

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.     

Chemokine receptor inhibitor,Antileukinate, suppressed ovalbumin-induced eosinophilic inflammation in the airway

Accumulating evidence suggests that eosinophils play an important role in the pathogenesis of allergic diseases. An eosinophil-active chemokine, eotaxin, and its receptor, C-C chemokine receptor 3, are particularly attractive as novel targets of immunological intervention for the disease. In this study, we examine the effects of a hexa-peptide (Ac-RRWWCR-NH(2)), Antileukinate, which we have previously defined as a potent inhibitor of CXC chemokine receptor 1 and 2, on eotaxin in vitro and in vivo. Antileukinate inhibited the binding of 125I-labeled human eotaxin to human eosinophils with an IC(50) of approximately 10 microM and eosinophil chemotaxis to human eotaxin was significantly inhibited by 10 microM of Antileukinate. We examined the effects of Antileukinate on eosinophil accumulation induced by intraperitoneal administration of murine eotaxin, and confirmed that Antileukinate is also active in the murine system. When Antileukinate was tested in ovalbumin-induced airway inflammation model in vivo, Antileukinate significantly inhibited eosinophil accumulation and allergen-induced increase in total protein in bronchoalveolar lavage fluids. Furthermore, Antileukinate suppressed fibrous thickening of submucosal tissue induced by chronic antigen challenge. These results suggest that eotaxin is involved in the pathogenesis of eosinophilic airway inflammation, and that Antileukinate may be a promising tool to control allergic diseases.     

Altered DNA methylation in leukocytes with trisomy 21

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.     

Shear Nanostructuring of Monoglyceride Organogels

The aim of the present research was to study the effect of shear on the crystallization behavior of monoglyceride organogels. To this end, organogels were prepared by mixing cod liver oil and saturated monoglycerides at 80°C and then crystallizing them at 20°C under shear rates ranging from 0 to 2,000 s⁻¹. The organogels were characterized using polarized light microscopy, Cryo-SEM, and X-ray diffraction. The rheological properties and the oil binding capacity of the different systems were also evaluated. Results obtained in this study showed that the introduction of shear during organogel formation greatly affects structure at the nano, micro, and macro levels. Solidification of the organogel under static conditions led to the formation of a strong gel network, with a high oil binding capacity. On the contrary, shear processing during crystallization led to the formation of a weak gel network with a low oil binding capacity.     

Mitochondrial carriers function as monomers

Mitochondrial carriers link biochemical pathways in the mitochondrial matrix and cytosol by transporting metabolites, inorganic ions, nucleotides and cofactors across the mitochondrial inner membrane. Uncoupling proteins that dissipate the proton electrochemical gradient also belong to this protein family. For almost 35 years the general consensus has been that mitochondrial carriers are dimeric in structure and function. This view was based on data from inhibitor binding studies, small-angle neutron scattering, electron microscopy, differential tagging/affinity chromatography, size-exclusion chromatography, analytical ultracentrifugation, native gel electrophoresis, cross-linking experiments, tandem-fusions, negative dominance studies and mutagenesis. However, the structural folds of the ADP/ATP carriers were found to be monomeric, lacking obvious dimerisation interfaces. Subsequently, the yeast ADP/ATP carrier was demonstrated to function as a monomer. Here, we revisit the data that have been published in support of a dimeric state of mitochondrial carriers. Our analysis shows that when critical factors are taken into account, the monomer is the only plausible functional form of mitochondrial carriers. We propose a transport model based on the monomer, in which access to a single substrate binding site is controlled by two flanking salt bridge networks, explaining uniport and strict exchange of substrates.     

Waves of dispersal in island-hopping Chondrina species (Gastropoda, Pulmonata, Chondrinidae)

The aim of this study was to unravel the historical biogeography of the speciose land snail genus Chondrina. To this end phylogenetic hypotheses were tested using mitochondrial DNA sequence data.Mitochondrial DNA sequences of the Cytochrome Oxidase subunit I region were obtained for 89 individuals, representing just over 70% of the extant Chondrina species. The extent of molecular genetic diversity and phylogeographical patterns were investigated by using neighbour joining, parsimony and bayesian methods for phylogeny reconstructions. The resulting data were used to infer historical biogeographical patterns for the genus Chondrina.The three phylogenetic methods yielded congruent topologies for the phylogeny reconstruction. Six clades were identified, each of which with at least one taxon that is known from the Iberian peninsula. The most parsimonious scenario indicates at least three waves of dispersal out of the Iberian peninsula into the North and East of Europe and Northern Africa.The phylogenetic relationships combined with the distributional patterns of the various species, indicate that only vicariance events cannot explain the actual situation. Apparently, separate waves of dispersal and subsequent speciation occurred, each time starting from the southwestern part of the present generic range. Until recently, this was obscured by repetitive cases of parallel or convergent evolution in shell characters, as became evident with the use of molecular methods.     

Percentage reward in an operant analogue to foraging

Pigeons (Columba livia) responding on an operant, conditioning analogue to foraging could either accept or reject a variable-interval 20-s schedule which always led to food or a variable-interval 5-s schedule which led to food on only a percentage of trials. The probability of accepting the certain alternative increased as the percentage of food trials for the uncertain alternative decreased. The probability of accepting the uncertain alternative increased with the percentage of food trials for this alternative. Subjects receiving all of their food in the experiment (‘closed economy’) and those requiring supplementation (‘open economy’) preferred whichever alternative provided the higher overall mean rate of reinforcement.