Human monocytes infected with Yersinia pestis express cell surface TLR9 and differentiate into dendritic cells

TLR9 recognizes DNA sequences containing hypomethylated CpG motifs and is a component of the innate immune system highly conserved during eukaryotic evolution. Previous reports suggested that the expression of TLR9 is restricted to plasmacytoid dendritic cells and B lymphocytes. Our results indicate that low levels of TLR9 are present on the cell surface of freshly isolated human monocytes, and expression is greatly increased by infection with Yersinia pestis. Enhanced cell surface TLR9 coincided with elevated levels of cytoplasmic TLR9 and recruitment of MyD88. Infected monocytes differentiated into mature dendritic cells, expressed IFN-alpha, and stimulated proliferative and cytotoxic T cell responses specific to Y. pestis. Furthermore, uninfected B cells and monocytes both increased cell surface TLR9, CD86, and HLA-DR in response to treatment with CpG-containing oligonucleotides, whereas cell surface TLR9 was down-modulated on infected dendritic cells by the addition of agonist oligonucleotide. Our results suggest that increased expression of TLR9 on the surface of infected cells may serve a role as an activation signal to other cells of the immune system.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

Diversity of lactic acid bacteria of the bioethanol process

Background  

Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.     

Catalysis of electron transfer by selenocysteine

Selenium is an essential element that is involved in biological redox processes. The electrode potentials of the selenocysteine half-reactions RSe(*) + e(-) --> RSe-, (RSeSeR)(*)(-) + e(-) --> 2 RSe(-), and RSeSeR + 2 e(-) --> 2 RSe(-) [E degrees' (pH 7)] are +0.43, +0.18, and -0.38 V, respectively, at pH 7. The spectra of RSe(*) and (RSeSeR)(*)(-) are characterized by absorption maxima at 460 nm (epsilon = 560 M(-)(1) cm(-)(1)) and 455 nm (epsilon = 7100 M(-)(1) cm(-)(1)), respectively. The bond dissociation energy of RSe-H has been calculated, and the value of 310 kJ/mol is in agreement with literature values. In comparison with the sulfur analogue cysteine, the more facile accessibility of the radical oxidation state is striking and may have biological implications, such as in mediation of one-electron- and two-electron-transfer processes, as illustrated by catalysis by selenocysteine of the electron transfer between dithiothreitol and benzyl viologen.     

Prenatal sex ratios and expression of sexually dimorphic traits in three snake species

Variation in intrauterine exposure to hormones associated with variation in the sex of litter mates has well-established and far-reaching effects on sexual development in some mammals. Research on this phenomenon in reptiles is scant, but suggests that lizards may follow the mammalian model whereas snakes may be affected differently. We examined sex-specific expression of four sexually dimorphic traits (tail length, head length, ventral scale count, swimming speed) in three species of snakes (Nerodia sipedon, Thamnophis sirtalis, T. sauritus) relative to litter sex ratios. We found little evidence that traits in either sex were masculinized or feminized in response to variation in litter sex ratio. The one significant result appeared best explained as a statistical artifact attributable to a single litter. Our results indicate that snakes are different from the one lizard studied to date. Unlike previous suggestions that prenatal hormonal mechanisms operate differently in snakes and lizards, however, the difference appears to be that development of sexually dimorphic traits in lizards is affected by litter sex ratios whereas in snakes it is not.     

Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1β. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.     

Mechanistic investigations of the liver toxicity of the free fatty acid receptor 1 agonist fasiglifam (TAK875) and its primary metabolites

For fasiglifam (TAK875) and its metabolites the substance‐specific mechanisms of liver toxicity were studied. Metabolism studies were run to identify a putatively reactive acyl glucuronide metabolite. In vitro cytotoxicity and caspase 3/7 activation were assessed in primary human and dog hepatocytes in 2D and 3D cell culture. Involvement of glutathione (GSH) detoxication system in mediating cytotoxicity was determined by assessing potentiation of cytotoxicity in a GSH depleted in vitro system. In addition, potential mitochondrial liabilities of the compounds were assessed in a whole‐cell mitochondrial functional assay. Fasiglifam showed moderate cytotoxicity in human primary hepatocytes in the classical 2D cytotoxicity assays and also in the complex 3D human liver microtissue (hLiMT) after short‐term treatment (24 hours or 48 hours) with TC₅₀ values of 56 to 68 µM (adenosine triphosphate endpoint). The long‐term treatment for 14 days in the hLiMT resulted in a slight TC₅₀ shift over time of 2.7/3.6 fold lower vs 24‐hour treatment indicating possibly a higher risk for cytotoxicity during long‐term treatment. Cellular GSH depletion and impairment of mitochondrial function by TAK875 and its metabolites evaluated by Seahorse assay could not be found being involved in DILI reported for TAK875. The acyl glucuronide metabolites of TAK875 have been finally identified to be the dominant reason for liver toxicity.     

Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88(-/-) mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1β, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1β production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.     

Rapid scavenging of peroxynitrous acid by monohydroascorbate

The reaction of peroxynitrous acid with monohydroascorbate, over the concentration range of 250 μM to 50 mM of monohydroascorbate at pH 5.8 and at 25°C, was reinvestigated and the rate constant of the reaction found to be much higher than reported earlier (Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. The kinetics of oxidation of L-ascorbic acid by peroxynitrite. Free Radic. Biol. Med. 18:85–92; 1995; Squadrito, G. L.; Jin, X.; Pryor, W. A. Stopped-flow kinetics of the reaction of ascorbic acid with peroxynitrite. Arch. Biochem. Biophys. 322:53–59; 1995). The new rate constants at pH 5.8 are k₁ = 1 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 500 s⁻¹ for 25°C and k₁ = 1.5 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 1 × 10³ s⁻¹ for 37°C. These values indicate that even at low monohydroascorbate concentrations most of peroxynitrous acid forms an adduct with this antioxidant. The mechanism of the reaction involves formation of an intermediate, which decays to a second intermediate with an absorption maximum at 345 nm. At low monohydroascorbate concentrations, the second intermediate decays to nitrate and monohydroascorbate, while at monohydroascorbate concentrations greater than 4 mM, this second intermediate reacts with a second monohydroascorbate to form nitrite, dehydroascorbate, and monohydroascorbate. EPR experiments indicate that the yield of the ascorbyl radical is 0.24% relative to the initial peroxynitrous acid concentration, and that this small amount of ascorbyl radicals is formed concomitantly with the decrease of the absorption at 345 nm. Thus, the ascorbyl radical is not a primary reaction product. Under the conditions of these experiments, no homolysis of peroxynitrous acid to nitrogen dioxide and hydroxyl radical was observed. Aside from monohydroascorbate's ability to “repair” oxidatively modified biomolecules, it may play a role as scavenger of peroxynitrous acid.