Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.     

Copper redistribution in murine macrophages in response to Salmonella infection

The movement of key transition metal ions is recognized to be of critical importance in the interaction between macrophages and intracellular pathogens. The present study investigated the role of copper in mouse macrophage responses to Salmonella enterica sv. Typhimurium. The copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) increased intracellular survival of S. Typhimurium within primary mouse BMM (bone-marrow-derived macrophages) at 24 h post-infection, implying that copper contributed to effective host defence against this pathogen. Infection of BMM with S. Typhimurium or treatment with the TLR (Toll-like receptor) 4 ligand LPS (lipopolysaccharide) induced the expression of several genes encoding proteins involved in copper transport [Ctr (copper transporter) 1, Ctr2 and Atp7a (copper-transporting ATPase 1)], as well as the multi-copper oxidase Cp (caeruloplasmin). Both LPS and infection with S. Typhimurium triggered copper accumulation within punctate intracellular vesicles (copper 'hot spots') in BMM as indicated by the fluorescent reporter CS1 (copper sensor 1). These copper hot spots peaked in their accumulation at approximately 18 h post-stimulation and were dependent on copper uptake into cells. Localization studies indicated that the copper hot spots were in discrete vesicles distinct from Salmonella containing vacuoles and lysosomes. We propose that copper hot spot formation contributes to antimicrobial responses against professional intracellular bacterial pathogens.     

Hepatitis B virus-induced lipid alterations contribute to natural killer T cell-dependent protective immunity

In most adult humans, hepatitis B is a self-limiting disease leading to life-long protective immunity, which is the consequence of a robust adaptive immune response occurring weeks after hepatitis B virus (HBV) infection. Notably, HBV-specific T cells can be detected shortly after infection, but the mechanisms underlying this early immune priming and its consequences for subsequent control of viral replication are poorly understood. Using primary human and mouse hepatocytes and mouse models of transgenic and adenoviral HBV expression, we show that HBV-expressing hepatocytes produce endoplasmic reticulum (ER)-associated endogenous antigenic lipids including lysophospholipids that are generated by HBV-induced secretory phospholipases and that lead to activation of natural killer T (NKT) cells. The absence of NKT cells or CD1d or a defect in ER-associated transfer of lipids onto CD1d results in diminished HBV-specific T and B cell responses and delayed viral control in mice. NKT cells may therefore contribute to control of HBV infection through sensing of HBV-induced modified self-lipids.     

Evidence of melanoma in wild marine fish populations

The increase in reports of novel diseases in a wide range of ecosystems, both terrestrial and marine, has been linked to many factors including exposure to novel pathogens and changes in the global climate. Prevalence of skin cancer in particular has been found to be increasing in humans, but has not been reported in wild fish before. Here we report extensive melanosis and melanoma (skin cancer) in wild populations of an iconic, commercially-important marine fish, the coral trout Plectropomus leopardus. The syndrome reported here has strong similarities to previous studies associated with UV induced melanomas in the well-established laboratory fish model Xiphophorus. Relatively high prevalence rates of this syndrome (15%) were recorded at two offshore sites in the Great Barrier Reef Marine Park (GBRMP). In the absence of microbial pathogens and given the strong similarities to the UV-induced melanomas, we conclude that the likely cause was environmental exposure to UV radiation. Further studies are needed to establish the large scale distribution of the syndrome and confirm that the lesions reported here are the same as the melanoma in Xiphophorus, by assessing mutation of the EGFR gene, Xmrk. Furthermore, research on the potential links of this syndrome to increases in UV radiation from stratospheric ozone depletion needs to be completed.     

Regulation of the endosomal SNARE protein syntaxin 7 by colony-stimulating factor 1 in macrophages

Colony-stimulating factor 1 (CSF-1) is the main growth factor controlling the development of macrophages from myeloid progenitor cells. However, CSF-1 also regulates some of the key effector functions of macrophages (e.g., phagocytosis and cytokine secretion). The endosomal SNARE protein syntaxin 7 (Stx7) regulates vesicle trafficking events involved in phagocytosis and cytokine secretion. Therefore, we investigated the ability of CSF-1 to regulate Stx7. CSF-1 upregulated Stx7 expression in primary mouse macrophages; it also upregulated expression of its SNARE partners Vti1b and VAMP8 but not Stx8. Additionally, CSF-1 induced the rapid serine phosphorylation of Stx7 and enhanced its binding to Vti1b, Stx8, and VAMP8. Bioinformatics analysis and results from experiments with kinase inhibitors suggested the CSF-1-induced phosphorylation of Stx7 was mediated by protein kinase C and Akt in response to phosphatidylinositol 3-kinase activation. Based on mutagenesis studies, CSF-1 appeared to increase the binding of Stx7 to its SNARE partners by inducing the phosphorylation of serine residues in the Habc domain and/or “linker” region of Stx7. Thus, CSF-1 is a key regulator of Stx7 expression and function in macrophages. Furthermore, the effects of CSF-1 on Stx7 may provide a mechanism for the regulation of macrophage effector functions by CSF-1.     

1,5-Hydride shift in Wolff-Kishner reduction of (20R)-3beta,20, 26-trihydroxy-27-norcholest-5-en-22-one: synthetic, quantum chemical, and NMR studies.

Heating (20R)-3beta,20,26-trihydroxy-27-norcholest-5-en-22-one (1) with hydrazine and KOH at 160 degrees C completely converted the steroid to a diastereoisomeric mixture of the new (20R,22RS)-27-norcholest-5-ene-3beta,20,22-triols (2). Exclusive formation of 2 suggests that the expected Wolff-Kishner reduction to a methylene group at the C-22 ketone in 1 was diverted to the C-26 position by a 1,5-hydride shift. All attempts under acid conditions failed to produce a C-22 phenyl hydrazone from 1. However, reaction of 1 was reacted with phenylhydrazine in hot KOH, gave the C-26 phenylhydrazone 4 as the sole product. Evidently, under alkaline conditions, first a hydride ion undergoes an intramolecular transfer from the C-26 CH(2)OH group to the C-22 ketone in 1, and then the phenylhydrazine traps the newly formed aldehyde. To examine this hypothesis, we constructed computer-simulated transition state models from quantum chemical calculations and then compared data from these models with NMR measurements of the reaction mixtures containing 2. The NMR data showed that the C-22 diastereoisomers of 2 are formed in a nearly 1:1 ratio exactly as predicted from the energy-optimized transition states, which were calculated for intramolecular 1,5-hydride shifts that produced each of the two C-22 diastereoisomers. Accordingly, these results support the hypothesis that an intramolecular 1,5-hydride shift mechanism promotes complete conversion of 1 to 2 under classical Wolff-Kishner reduction conditions.     

Structural studies on proteoglycan from human chondrosarcoma.

Proteoglycan was isolated from a human chondrosarcoma which contained all glycosaminoglycans found in articular cartilage. Proteoglycans extracted by associative (67% of total uronate) and subsequent dissociative (27% of total uronate) solvents were identical as assessed by chromatography on Sepharose 2B (K_av 0.43), electrophoresis on acrylamideagarose gels, and in their ability to bind to hyaluronate. In addition there were no differences in chondroitin sulfate size, ratio of chondroitin 4- to 6-sulfate, or in size or form of keratan sulfate present. Two forms of keratan sulfate were identified following treatment with alkaline borohydride: A larger species (～-23 monosaccharides) was isolated from the keratan sulfate-enriched region only; a smaller oligosaccharide (～-13 monosaccharides) was recovered from all peptidoglycans released by trypsin, chymotrypsin treatment.     

Repair of 3-methyladenine and 7-methylguanine in nuclear DNA of Chlamydomonas: Requirement for protein synthesis

The removal of 3-methyladenine and 7-methylguanine from nuclear DNA was determined following exposure of Chlamydomonas reinhardi to methyl methanesulfonate (MMS). The amount of 3-methyladenine in DNA was determined using an extract from Micrococcus luteus that has a 3-methyladenine-DNA glycosylase. The amount of 7-methylguanine was estimated by heating the DNA for 30 min at 70° followed by alkaline hydrolysis of the resulting apurinic sites. The molecular weight of the DNA was determined using alkaline sucrose gradients. The 3-methyladenine is removed with a half-life of 2–3 h whereas the 7-methylaguanine is removed with a half-life of 10–12 h. The rate of removal of the 7-methylguanine is more than an order of magnitude faster than the estimated non-enzymatic hydrolysis rate indicating the probability of enzymatic repair. Addition of cycloheximide immediately after MMS treatment inhibits the removal of 3-methyladenine and 7-methylguanine from DNA. If cycloheximide is added 1.5 h after treatment with MMS, there is much less inhibition of the removal of 3-methyladenine. These results are interpreted to mean that MMS induces the synthesis of 1 or more proteins that are required for the repair of 3-methyladenine from Chlamydomonas DNA.