Bovine brain microsomal CDP-diacylglycerol synthetase: solubilization and properties.

CDP-diacylglycerol(DAG) synthetase (EC 2.7.7.41) has been solubilized from bovine brain microsomes by the detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate). Optimal solubilization with 1.5% CHAPS yielded 55-60% of the synthetase activity. The effect of CHAPS on the enzyme was biphasic inhibiting at 0.3% and giving maximal activity at 0.5% (the concentration used for all assays). The solubilized, but not the microsomal enzyme is activated by phosphatidylcholine (PC) and strongly inhibited by cardiolipin and lysoPC. Strong inhibition by N-ethylmaleimide, 5,5'-dithio-bis (2-nitrobenzoic acid) and p-chloromercuribenzoate supported a sulfhydryl requirement for the enzyme. Phosphatidic acid (PA) from egg lecithin and 1-stearoyl,2-arachidonoyl PA were preferred substrates for the microsomal synthetase. Solubilized synthetase showed selectivity for the latter PA which is consistent with this enzyme functioning to help form the preponderant 1-stearoyl,2-arachidonoyl species of phosphatidylinositol. Further attempts to purify the synthetase were unsuccessful. All findings suggested the enzyme exists as an unstable complex.     

Inhibition of NF-kappaB activation reduces the tissue effects of transgenic IL-13

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.     

Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa

Airway mucosal dendritic cells (AMDC) and other airway APCs continuously sample inhaled Ags and regulate the nature of any resulting T cell-mediated immune response. Although immunity develops to harmful pathogens, tolerance arises to nonpathogenic Ags in healthy individuals. This homeostasis is thought to be disrupted in allergic respiratory disorders such as allergic asthma, such that a potentially damaging Th2-biased, CD4(+) T cell-mediated inflammatory response develops against intrinsically nonpathogenic allergens. Using a mouse model of experimental allergic airways disease (EAAD), we have investigated the functional changes occurring in AMDC and other airway APC populations during disease onset. Onset of EAAD was characterized by early and transient activation of airway CD4(+) T cells coinciding with up-regulation of CD40 expression exclusively on CD11b(-) AMDC. Concurrent enhanced allergen uptake and processing occurred within all airway APC populations, including B cells, macrophages, and both CD11b(+) and CD11b(-) AMDC subsets. Immune serum transfer into naive animals recapitulated the enhanced allergen uptake observed in airway APC populations and mediated activation of naive allergen-specific, airway CD4(+) T cells following inhaled allergen challenge. These data suggest that the onset of EAAD is initiated by enhanced allergen capture and processing by a number of airway APC populations and that allergen-specific Igs play a role in the conversion of normally quiescent AMDC subsets into those capable of inducing airway CD4(+) T cell activation.     

Frequencies of single nucleotide polymorphisms in genes regulating inflammatory responses in a community-based population

Background

Researchers may embark on a genome-wide association study before fully investigating candidate regions which have been reported to produce evidence to suggest that they harbour susceptibility loci. If the genome wide study had not been carried out then results which demonstrated only modest statistical significance from candidate regions would be judged to be of interest and would stimulate further investigation. However if hundreds of thousands of markers are typed then inevitably very large numbers of such results will occur by chance and those from candidate regions may attract no special attention.

Results

An approach is proposed in which differential treatment is afforded to markers from candidate regions and from those that are routinely typed in the context of a genome wide scan. Different prior probabilities are assigned to the two types of marker. A likelihood ratio is derived from the reported p value for each marker, calculated as LR = e^{chiinv(1,p)/2}, and the posterior odds in favour of a true positive association are obtained. These odds can be used to rank the markers with a view to suggesting the regions in which further genotyping is indicated. We suggest that prior probabilities be specified such that a candidate marker significant at p = 0.01 and a routine marker significant at p = 0.00001 will yield similar values for the posterior odds. We show that this can be achieved by setting a value for prior probability of association to 0.1 for candidate markers and to 0.00018 for routine markers.

Conclusion

It is essential that formal procedures be adopted in order to avoid modestly positively results from candidate regions being swamped by the huge number of nominally significant results which will be obtained when very many markers are genotyped. Software to carry out the conversion from p values to posterior odds is available from http://www.mds.qmul.ac.uk/statgen/grpsoft.html.     

Neuroserpin polymorphisms and stroke risk in a biracial population: the stroke prevention in young women study

Background

Neuroserpin, primarily localized to CNS neurons, inhibits the adverse effects of tissue-type plasminogen activator (tPA) on the neurovascular unit and has neuroprotective effects in animal models of ischemic stroke. We sought to evaluate the association of neuroserpin polymorphisms with risk for ischemic stroke among young women.

Methods

A population-based case-control study of stroke among women aged 15–49 identified 224 cases of first ischemic stroke (47.3% African-American) and 211 age-matched control subjects (43.1% African-American). Neuroserpin single nucleotide polymorphisms (SNPs) chosen through HapMap were genotyped in the study population and assessed for association with stroke.

Results

Of the five SNPs analyzed, the A allele (frequency; Caucasian = 0.56, African-American = 0.42) of SNP rs6797312 located in intron 1 was associated with stroke in an age-adjusted dominant model (AA and AT vs. TT) among Caucasians (OR = 2.05, p = 0.023) but not African-Americans (OR = 0.71, p = 0.387). Models adjusting for other risk factors strengthened the association. Race-specific haplotype analyses, inclusive of SNP rs6797312, again demonstrated significant associations with stroke among Caucasians only.

Conclusion

This study provides the first evidence that neuroserpin is associated with early-onset ischemic stroke among Caucasian women.     

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.