A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.     

Adenosine Stimulates Cone Photoreceptor Myoid Elongation via an Adenosine A2-Like Receptor

In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment-cone outer segment preparations (CIS-COS) in vitro. Agonist/antagonist potency profiles indicate that adenosine's effect on cone movement is mediated by an A2-like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS-COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory "dark signal" influencing photoreceptor metabolism and function in the fish retina.     

Nitric oxide inhibits prooxidant actions of uric acid during copper-mediated LDL oxidation

Interactions between uric acid and physiologically relevant fluxes of nitric oxide ((?)NO) during copper-mediated low-density lipoprotein (LDL) oxidation were evaluated. In the absence of (?)NO, a dual pro- and antioxidant action of uric acid was evident: low concentrations of uric acid enhanced lipid oxidation and alpha-tocopherol consumption, while its protective role was observed at higher concentrations. The prooxidant effects of uric acid were mostly related to its copper-reducing ability to form Cu(+), an initiator of lipid oxidation processes. While the prooxidant action of uric acid was completely inhibited by (?)NO, the antioxidant action of (?)NO was slightly counterbalanced by uric acid. Enhancement of alpha-tocopherol consumption by uric acid was inhibited in the presence of (?)NO while additive antioxidant effects between (?)NO and uric acid were observed in conditions where uric acid spared alpha-tocopherol. Altogether, these results suggest that in the artery wall, the (?)NO/uric acid pair may exert antioxidant actions on LDL, even if increased amounts of redox active copper were available at conditions favoring prooxidant activities of uric acid.     

Respiratory syncytial virus infection activates STAT signaling in human epithelial cells

Acute respiratory syncytial virus (RSV) infection causes airway inflammation and exacerbates asthma, but the mechanism of inflammation is poorly understood. The role of the STAT-signaling pathway in RSV infection in epithelial cells was examined in this study. DNA microarray analyses of RSV-infected human alveolar type II (A549) epithelial cells identified several genes whose expression was altered from -5.5 to +56.4-fold. Four of the highly expressed genes contained STAT-binding elements. In A549 and normal human bronchial epithelial cells (NHBE), RSV induced phosphorylation and nuclear translocation of STAT-1alpha that was abrogated when RSV attachment was blocked. Treatment with a JAK-2 inhibitor or transfection with dominant-negative STAT-1alpha blocked STAT-1alpha activation and RSV infection. RSV also activated STAT-3 and IL-6 specific antibodies blocked this activation. Thus, activation of the STAT-1alpha and STAT-3 pathways play a role in RSV infection.     

Extramitochondrial localization of NADH-fumarate reductase in trypanosomatids

Trypanosoma brucei procyclic trypomastigotes and T. cruzi epimastigotes (both Tulahuen and Y strains) were permeabilized by incubation with increasing amounts of digitonin, causing enzymes to be released from different intracellular compartments. After 10 min incubation with digitonin, the cells were centrifuged and the activity of marker enzymes (aspartate-dependent malic enzyme for cytoplasm, hexokinase for glycosomes and either isocitrate dehydrogenase or citrate synthase for mitochondria) was analyzed in the supernatant. The results were compared with the release of NADH-fumarate reductase in order to determine if this enzyme was preferentially released with a specific intracellular marker. Fumarate reductase was released at lower digitonin concentration than those required to either release isocitrate dehydrogenase or citrate synthase. Similarly, Leishmania donovani promastigotes (S-2 strain) were exposed to a single concentration of digitonin (200 micro M) but in this case we monitored the release of fumarate reductase and hexokinase, while monitoring the mitochondrial membrane potential (using safranine O). Again, substantial fumarate reductase and hexokinase activities were released without loss of mitochondrial membrane potential indicating that part of the enzyme was released while the inner mitochondrial membrane remained intact. These results suggest that, in the three species of trypanosomatids the enzyme fumarate reductase is, at least in part, located outside the mitochondrial matrix.     

Characterization of linoleic acid nitration in human blood plasma by mass spectrometry

Nitric oxide (*NO) is a pervasive free radical species that concentrates in lipophilic compartments to serve as a potent inhibitor of lipid and low-density lipoprotein oxidation processes. In this study, we synthesized, characterized, and detected nitrated derivatives of linoleic acid (18:2) in human blood plasma using high-pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry. While the reaction of nitronium tetrafluoroborate with 18:2 presented ions with a mass/charge (m/z) ratio of 324 in the negative ion mode, characteristic of nitrolinoleate (LNO(2)), the reaction of nitrite (NO(2)(-)) with linoleic acid hydroperoxide yielded nitrohydroxylinoleate (LNO(2)OH, m/z 340). Further analysis by MS/MS gave a major fragment at m/z 46, characteristic of a nitro group (-NO(2)) present in the parent ion. This was confirmed by using [(15)N]O(2), which gave products of m/z 325 and 341, that after fragmentation yielded a daughter ion at m/z 47. Moreover, a C-NO(2) structure was also demonstrated in LNO(2)OH by nuclear magnetic resonance spectroscopy ((15)N NMR, delta 375.9), as well as by infrared analysis in both LNO(2)OH (nu(max) = 3427, 1553, and 1374 cm(-1)) and LNO(2) (nu(max) = 1552 and 1373 cm(-1)). Stable products with m/z of 324 and 340, which possessed the same chromatographic characteristics and fragmentation pattern as synthesized standards, were found in human plasma of normolipidemic and hyperlipidemic donors. The presence of these novel nitrogen-containing oxidized lipid adducts in human plasma could represent "footprints" of the antioxidant action of *NO on lipid oxidation and/or a pro-oxidant and nitrating action of *NO-derived species.     

Psychosocial assessment for victims of violence in Peru: the importance of local participation

This paper describes a pilot study assessing the psychosocial impact of political violence in the Peruvian Andes, utilizing a collaborative approach with local professionals and communities. The study team prioritized dialogue and information exchange with the local professional community and villagers participating in the assessment in order to raise awareness of psychosocial issues and provide education and support. Participation in the pilot study had positive therapeutic effects for villagers, and inspired ongoing discussion groups to address psychosocial problems in communities. This paper also describes a psychosocial assessment strategy utilizing qualitative methods and an adaptation of the Harvard Trauma Questionnaire in collaboration with Andean villagers. Usefulness and limitations of the data will be reviewed, in terms of cultural and context relevance, usefulness for informing interventions, and comparisons with ethnographic methodologies and other survey instruments.