20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide: photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid receptor

Endothelium-derived epoxyeicosatrienoic acids (EETs) relax vascular smooth muscle by activating potassium channels and causing membrane hyperpolarization. Recent evidence suggests that EETs act via a membrane binding site or receptor. To further characterize this binding site or receptor, we synthesized 20-iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide (20-I-14,15-EE8ZE-APSA), an EET analogue with a photoactive azido group. 20-I-14,15-EE8ZE-APSA and 14,15-EET displaced 20-(125)I-14,15-epoxyeicosa-5(Z)-enoic acid binding to U937 cell membranes with K(i) values of 3.60 and 2.73 nM, respectively. The EET analogue relaxed preconstricted bovine coronary arteries with an ED(50) comparable to that of 14,15-EET. Using electrophoresis, 20-(125)I-14,15-EE8ZE-APSA labeled a single 47 kDa band in U937 cell membranes, smooth muscle and endothelial cells, and bovine coronary arteries. In U937 cell membranes, the 47 kDa radiolabeling was inhibited in a concentration-dependent manner by 8,9-EET, 11,12-EET, and 14,15-EET (IC(50) values of 444, 11.7, and 8.28 nM, respectively). The structurally unrelated EET ligands miconazole, MS-PPOH, and ketoconazole also inhibited the 47 kDa labeling. In contrast, radiolabeling was not inhibited by 8,9-dihydroxyeicosatrienoic acid, 5-oxoeicosatetraenoic acid, a biologically inactive thiirane analogue of 14,15-EET, the opioid antagonist naloxone, the thromboxane mimetic U46619, or the cannabinoid antagonist AM251. Radiolabeling was not detected in membranes from HEK293T cells expressing 79 orphan receptors. These studies indicate that vascular smooth muscle, endothelial cells, and U937 cell membranes contain a high-affinity EET binding protein that may represent an EET receptor. This EET photoaffinity labeling method with a high signal-to-noise ratio may lead to new insights into the expression and regulation of the EET receptor.     

Diverse effects of residues 74–78 in ribosomal protein S12 on decoding and antibiotic sensitivity

Ribosomal protein S12 plays key roles in the ribosome’s response to the error-promoting antibiotic streptomycin and in modulating the accuracy of translation. The discovery that substitutions at His76 in S12, distant from the streptomycin binding site, conferred streptomycin resistance in the thermophilic bacterium Thermus thermophilus prompted us to make similar alterations in the S12 protein of Escherichia coli. While, none of the E. coli S12 mutations confers streptomycin resistance, they all have distinct effects on the accuracy of translation. In addition, a subset of the S12 alterations renders the cells hypersensitive to fusidic acid, an inhibitor of the translocation step of translation. These results indicate that the His 76 region of ribosomal protein S12 plays key roles in tRNA selection and translocation steps of protein synthesis, consistent with its interaction with elongation factors EF-Tu and EF-G, as deduced from structural studies of ribosomal complexes.     

A Toolbox for Tuberculosis (TB) Diagnosis: An Indian Multi-Centric Study (2006-2008); Evaluation of Serological Assays Based on PGL-Tb1 and ESAT-6/CFP10 Antigens for TB Diagnosis

Background

The aim of this multi-centric prospective study in India was to assess the accuracy of a serological test as an additional tool for diagnosing active tuberculosis (ATB). In particular, an assay based on ELISA using a phenolic glycolipid (PGL-Tb1) or a fusion protein (ESAT-6/CFP10) was compared to the tuberculin skin test (TST) and the microbiological results according to HIV status.

Methods

Individuals with and without ATB and HIV infection were enrolled. Serology and TST results were analyzed per se and in combination with the microbiological data.

Results

Among the 778 ATB patients, 102 were HIV-infected, 316 HIV-uninfected and 360 had an HIV-unknown status. Of the 945 non-ATB subjects, 559 were at low risk (community adults) and 386 at high risk of M. tuberculosis exposure. Among those with ATB, the sensitivity of ELISA-PGL-Tb1 for ATB was higher than that of ELISA-ESAT-6/CFP10, both in HIV-infected (72.3% versus 63.7%, p = 0.29) and HIV-uninfected/HIV-unknown groups (40.5% versus 28.6%; p<0.0001), whereas the specificity was around 91% for both tests. Sensitivity for ATB increased when the results of the two ELISA were combined, reaching 75.5% in the HIV-infected and 50.9% in the group of HIV-uninfected/HIV-unknown ATB, with a significant decrease of the global specificity (83.9%). Analyzing the ELISA results with the microbiological results, we observed that the sensitivity of both serology tests was independent of the ATB patients'' smear microscopy (SM) status and grade. Combining the results of SM with both ELISA, the detection of ATB patients significantly increased (p<0.0001), particularly in those with extrapulmonary TB (up to 45.1%) or HIV infection (up to 83.3%). No significant association was observed between TST and serology results.

Conclusions

In this prospective multi-centric study, the combination of two rapid tests, such as SM and serology, might be useful in detecting ATB, especially in HIV-infected patients.     

Nutritional supremacy of pearl- and foxtail millets: assessing the nutrient density,protein stability and shelf-life of flours in millets and cereals for developing nutri-stable foods

Journal of Plant Biochemistry and Biotechnology - Flour of millets is nutritionally better, as compared to cereals, but has problem of low shelf-life. Here, we analyzed the nutritional density, and...     

Arrestin-2 interacts with the ubiquitin-protein isopeptide ligase atrophin-interacting protein 4 and mediates endosomal sorting of the chemokine receptor CXCR4

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.     

Modulation of Decoding Fidelity by Ribosomal Proteins S4 and S5

Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.     

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic'' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.     

Long term immune responses to pandemic influenza A/H1N1 infection in solid organ transplant recipients

In solid organ transplant (SOT) recipients it is unknown if natural infection with influenza confers protection from re-infection with the same strain during the next influenza season. The purpose of this study was to determine if infection with pandemic influenza A/H1N1 (pH1N1) resulted in a long-term immunologic response. Transplant recipients with microbiologically proven pH1N1 infection in 2009/2010 underwent humoral and cell-mediated immunity (CMI) testing for pH1N1 just prior to the next influenza season. Concurrent testing for A/Brisbane/59/2007 was done to rule-out cross-reacting antibody. We enrolled 22 adult transplant patients after pH1N1 infection. Follow up testing was done at a median of 7.4 months (range 5.8-15.4) after infection. After excluding those with cross-reactive antibody, 7/19 (36.8%) patients were seroprotected. Detectable pH1N1-specific CD4+ and CD8+ interferon-γ producing T-cells were found in 11/22 (50%) and 8/22 (36.4%) patients respectively. Humoral immunity had a significant correlation with a CD4 response. This is the first study in transplant patients to evaluate long-term humoral and cellular response after natural influenza infection. We show that a substantial proportion of SOT recipients with previous pH1N1 infection lack long-term humoral and cellular immune responses to pH1N1. These patients most likely are at risk for re-infection.