Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe

[(3)H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([(3)H]HBP) was synthesized and used to photoaffinity label P450(Cam). The imidazole moiety of HBP anchors the compound in the P450(Cam) active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450(Cam) from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450(Cam). Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B'/C loop region of P450(Cam). The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.     

RGS2 inhibits the epithelial Ca2+ channel TRPV6

The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) entry pathway in the process of active Ca(2+) (re)absorption. By yeast two-hybrid and glutathione S-transferase pulldown analysis we identified RGS2 as a novel TRPV6-associated protein. RGS proteins determine the inactivation kinetics of heterotrimeric G-protein-coupled receptor (GPCR) signaling by regulating the GTPase activity of G(alpha) subunits. Here we demonstrate that TRPV6 interacts with the NH(2)-terminal domain of RGS2 in a Ca(2+)-independent fashion and that overexpression of RGS2 reduces the Na(+) and Ca(2+) current of TRPV6 but not that of TRPV5-transfected human embryonic kidney 293 (HEK293) cells. In contrast, overexpression of the deletion mutant DeltaN-RGS2, lacking the NH(2)-terminal domain of RGS2, in TRPV6-expressing HEK293 cells did not show this inhibition. Furthermore, cell surface biotinylation indicated that the inhibitory effect of RGS2 on TRPV6 activity is not mediated by differences in trafficking or retrieval of TRPV6 from the plasma membrane. This effect probably results from the direct interaction between RGS2 and TRPV6, affecting the gating properties of the channel. Finally, the scaffolding protein spinophilin, shown to recruit RGS2 and regulate GPCR-signaling via G(alpha), did not affect RGS2 binding and electrophysiological properties of TRPV6, indicating a GPCR-independent mechanism of TRPV6 regulation by RGS2.     

Antibiotic translocation through membrane channels: temperature-dependent ion current fluctuation for catching the fast events

Temperature-dependent facilitated permeation of antibiotics through membrane channels was investigated. Here we reconstituted single OmpF trimers from the outer membrane of Escherichia coli (E. coli) into a planar lipid bilayer. The penetration of ampicillin through OmpF causes fluctuation in the ion current, and analysis of the fluctuations at different temperatures allows us to determine the mode of permeation. The residence time of the drug inside the channel decays strongly with temperature, reaching the resolution limit of the instrument at 30°C. The number of events increases exponentially with temperature up to 30°C and then gradually decreases as temperature increases. At room temperature, we observe about 25 events per second per monomer of the trimeric channel and an extrapolation to 37°C gives roughly 50 events. The activation energy for ampicillin translocation through OmpF is estimated to be around 13 kT. Temperature-dependent study gives new insights into the faster translocation of small substrates through biological nanopores.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

Effect of various normalization methods on Applied Biosystems expression array system data

Background  

DNA microarray technology provides a powerful tool for characterizing gene expression on a genome scale. While the technology has been widely used in discovery-based medical and basic biological research, its direct application in clinical practice and regulatory decision-making has been questioned. A few key issues, including the reproducibility, reliability, compatibility and standardization of microarray analysis and results, must be critically addressed before any routine usage of microarrays in clinical laboratory and regulated areas can occur. In this study we investigate some of these issues for the Applied Biosystems Human Genome Survey Microarrays.     

Synthesis and absolute configuration assignment of 5-amino-1,3,5-triphenyl-pentane-1,3-diol stereoisomers

Starting from 2,4,6-triphenylpyrylium perchlorate, 5-amino-1,3,5-triphenyl-pentane-1,3-diol stereoisomers 4 were obtained in a simple two-step synthesis: reaction with hydroxylamine, and reduction with LAH of the resulting 2-isoxazoline ketone derivative 2. The eight stereoisomers of 4 were separated in a single shot on a chiral stationary phase cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD-H). The absolute configuration of the title compounds, intermediate 2-isoxazoline ketone 2 and isoxazoline alcohol derivative 3 were determined using a combination of diastereoselective synthesis, affiliation of the sign in chemical interconversion method, and X-ray determination. 2-Isoxazoline ketone 2 enantiomers and isoxazoline alcohol 3 enantiomers were obtained by chiral HPLC on Chiralpak AD column. 2-Isoxazoline ketone 2 enantiomers can be racemized via a retro Michael addition.     

Role of disulfide bond formation in the folding and assembly of the envelope glycoproteins of a pestivirus

Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, closely related to, and used as a surrogate model for the hepatitis C virus. Its envelope contains the E1 and E2 glycoproteins, disulfide linked into homo- and heterodimers. In this study, we investigate the role of disulfide bond formation in the folding, assembly, and stability of BVDV glycoproteins. We provide molecular evidence that intact disulfide bonds are critical for the acquirement of a stable conformation of E2 monomers. Forcing the E2 glycoproteins to adopt a reduced conformation either co- or post-translationally before assembly into dimers, determines their misfolding and degradation by proteasome. In contrast, dimerization of E2 glycoproteins results in a conformation resistant to reducing agents and degradation. Furthermore, inhibition of the ER-alpha-mannosidase activity leads to impairment of misfolded E2 degradation, demonstrating the involvement of this enzyme in targeting viral proteins towards proteasomal degradation.     

Spontaneous ribosome bypassing in growing cells

Translating ribosomes can pass through a stretch of messenger RNA without translating and resume protein chain elongation after the bypassed region. We previously investigated the stimulation of bypassing when the codon in the ribosome [corrected] A-site called for an aminoacyl-tRNA species in short supply. Here, we investigate bypassing in unstarved, growing cells. A collection of lacZ bypass reporters was constructed with nearly all the sense codons as the "takeoff site", each with its matched landing site 16 nucleotides downstream in the beta-galactosidase reading frame. Beta-galactosidase [corrected] synthesis in unstarved cells carrying these reporters was found to vary over a large range. The takeoff sites UUU and AGG yielded unusually high enzyme activities, sufficient for protein sequence analysis; in these cases, sequencing (by Edman degradation or by mass spectrometry) confirmed that the synthesis of lacZ protein occurred through the 16 nt bypass from takeoff to landing site. Thus, bypassing occurs spontaneously under normal conditions, and is not limited to the pathology of amino acid starvation. Indirect evidence suggests that most of the lower enzyme activities of the rest of the collection also reflects bypassing. Another collection of reporters was made with [corrected] various triplets in the A-site [corrected] the codon immediately following a UUC [corrected] takeoff triplet. Spontaneous bypassing in representatives of this collection varied roughly inversely with the abundance of the tRNA encoded at the A-site. For two A-site codons tested, introduction of additional copies of the relevant tRNA gene on a second plasmid reduced spontaneous bypassing. We conclude that any pause with the A-site empty stimulates bypassing. From the P-site and A-site effects on bypassing, we estimated the average frequency of ribosome takeoff; from this, we calculate that the probability that a ribosome will succeed in translating the entire lacZ coding sequence is about 0.73, in agreement with earlier, independent estimates.