Regulation of translation is required for dendritic cell function and survival during activation

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response.     

Deconvolution as a novel approach to analyze moment-to-moment free fatty acid release

Objective: Recent studies have shown that free fatty acid (FFA) release is pulsatile and that this pattern is controlled by the sympathetic nervous system. It is, then, necessary to understand and characterize adipose tissue lipolysis to elucidate its effect on metabolism. In this study, we introduce deconvolution as a method to detect and quantify pulsatile FFA release. Research Methods and Procedures: Octanoate, a medium‐chain fatty acid, was infused in male mongrel dogs (n = 7) to mimic the pulsatile appearance of plasma FFAs. Deconvolution analysis was used to reconstruct the number and timing of infused octanoate pulses from plasma FFA concentrations. Results: Deconvolution analysis was able to reconstruct the exogenously infused pulses of octanoate used to mimic pulsatile appearance of FFAs (pulse frequency, 8 per hour; interpulse interval, 7 minutes). However, determination of pulse mass was less accurate (1.0 ± 0.0 vs. 0.54 ± 0.1 mM). The addition of varying levels of Gaussian noise to non‐oscillatory FFA time series did not lead to detection of extraneous FFA pulses. However, goodness of fit declined with increasing variability. Discussion: These results support the use of deconvolution as an accurate approach to determine the temporal sequence of endogenous FFA release.     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.     

Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon.

We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.     

Cardiolipin interacts with beta‐2‐glycoprotein I and forms an open conformation–Mechanisms analyzed using hydrogen/deuterium exchange

Beta‐2‐glycoprotein I (β₂GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β₂GPI can be recognized by the anti‐β₂GPI antibody. Here, we prepared the anionic di‐oleoyl‐phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β₂GPI. The conformational changes of β₂GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21–27 significantly increased after β₂GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21–27 from the ring conformation. After β₂GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1–20, 21–27, 196–205, 273–279 and 297–306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70–86, 153–162, 191–198, 196–205 and 273–279 indicated β₂GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β₂GPI at sequences 21–27 and forms an intermediate conformation after 10 min of interaction. After a complete protein–lipid interaction, high negatively charged CL membrane induced a major conformation transition of β₂GPI.