with 1 mM rosiglitazone (Enzo Life Sciences), 1 mM GW1929 (Tocris Bioscience) or the vehicle as indicated in the figures. Forty- eight hours post transfection, cells were lysed in 1 ml lysis buffer (6 M guanidinium HCl, 0.1 M sodium phosphate buffer pH 8.0, 0.05% Tween 20, 20 mM imidazole), and His-SUMO modified proteins were isolated by incubation with 20 ml of Ni-NTA magnetic agarose beads (Qiagen) for 16 hours at 4 uC. Beads were washed three times each with 750 ml buffer A (8 M urea, 0.1 M sodium phosphate buffer pH 8.0, 0.05% Tween 20, 20 mM imidazole) and buffer B (8 M urea, 0.1 M sodium phosphate buffer pH 6.4, 0.05% Tween 20, 20 mM imidazole). After a final washing step with phosphate buffered saline, the beads were boiled in 50 ml SDS sample buffer. Proteins were separated by SDS- PAGE and subsequently transferred on an Immobilon-P mem- brane (Millipore) for chemiluminescence or on an Immobilon-FL membrane (Millipore) for fluorescence detection according to the manufacturer’s instructions. Primary and secondary antibody incubations were carried out in 1% skim milk for 1 hour each at room temperature. The rat anti-HA antibody (3F10, Roche) was used for chemiluminescence (1:2000 dilution) and for fluorescence (1:1000 dilution) detection of HA-PPARc1 proteins. The anti- FLAG M2 (Sigma), 1:1000, antibody was used for detection of FLAG-PPARc (1-256) and FLAG-PPARc (247-475). Visualiza- tion of immunoblots by chemiluminescence was performed with horseradish peroxidase-coupled anti-rat or anti-mouse antibodies (GE Healthcare Life Science, 1:15,000) followed by incubation with the Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore). The IRDye 680-labeled anti-rat secondary antibody (LI-COR Biosciences, 1:2000) was used for quantitative fluorescent detection with the Odyssey Infrared Imager (LI-COR Biosciences).
Recent studies have reported that macrophages are a major component in the immune response involved in allograft rejection [35,36]. CAM accelerate the immune response, whereas AAM have repair and anti-inflammatory abilities . Moreover, PPARc agonists skewed monocytes toward AAM polarization . We have observed that without PPARc in T cells, PPARc agonists cannot exert protective function on macrophages. In additional studies, we investigated macrophage polarization in T cell-PPARc ko mice. Previous studies have indicated that IL-4 and IL-13 binding to IL-4Ra and IL-13Ra1 resulted in downstream phosphorylation of STAT6, thereby causing macrophage polar- ization [38–40]. More recently, Szanto et al. proposed that PPARc plays a critical role in these path ways because IL-4 signaling activates PPARc through STAT6 interactions with PPARc and promotes their binding to PPRE . Indeed, the IL- 4/13-STAT6-KLF4-PPARc axis is regarded as an essential regulator of CAM/AAM polarization and function . In our experiments, the ratio of Th2 decreased, and Th2-related cytokine production was reduced. The AAM markers Arg1 and Mrc1 were down-regulated after T cell specific knockout of PPARc. Additionally, Tiemessen et al indicated that Treg have the ability Figure 6. PPARc ko Treg fail to induce AAM in a monocyte and T cell coculture. CD4+CD25+ T cells were isolated from the spleens of T cell- PPARc ko mice (ko) and WT littermates (wt) and cocultured with CD11b+ monocytes from PBMC C57BL/6 mice. The transcription level of iNOS, Arg1,
PPAR-c activation has recently been shown to be a rational and effective strategy against cerebral I/R injury [5–8]. PPAR-c agonists efficiently protect against cerebral I/R in rats. The mechanisms of neuroprotection following PPAR-c activation include antioxidative properties and anti-inflammatory effect. Recent studies suggest that ligand-activated PPAR-c controls apoptosis and contributes to neuroprotection [9,10,33,34]. Figure 8. Protective effects of 3-mehtyladenine (3-MA) following cerebral I/R injury. 3-MA (60 mg) solutions were injected icv immediately before reperfusion. (A) The changes of LC3 after the treatment of 3-MA. 3-MA significantly decreased LC3-II levels at 24 h after I/R. Effect of 3-MA on infarct volumes (B) and neurological deficits (C). 3-MA significantly reduced the infarct volume, and ameliorate the neurological symptoms at 24 h after I/R. #
Here, we crystallized PPARc LBD in a form that diffracts to relatively high resolution in the absence of exogenous ligands. The structure resembles previous liganded and unliganded PPARs [4,18] but close investigation reveals three saturated medium chain fatty acids (MCFAs) occupy the LBP at the same time and mass spectroscopic analysis suggests that these are predominantly nonanoic acid (NA, C9) with a smaller amount of octanoic acid (OA, C8). C8–C10 MCFAs are PPARc essentially partial agonists, but exhibit assay-specific variations in activity relative to TZDs and MCFAs that bind PPARc block TZD-dependent adipogen- esis. A recent paper also revealed that a C10 MCFA acts as a modulating ligandof PPARc, but this group found a single molecule of C10 binds the pocket and rationalized partial agonist activity in terms of weak H12 stabilization . Our X-ray crystal structure B-factor analysis coupled to molecular dynamics (MD) simulations  suggests that diverse agonist/partial agonist behaviors may be linked to the tripartite MCFA binding mode and raise the intriguing possibility that selective PPAR modulators with useful context-selective properties may be identified among natural products. We discuss the possibility that MCFAs are natural PPAR ligands.
Lysophosphatidic acid (LPA) is an agonist for peroxisomeproliferatoractivatedreceptor-c (PPARc). Although glycerol-3- phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARc activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARc target, was 2-fold higher. Transactivation assays showed that PPARc activity was higher in the cells that overexpressed GPAT1. PPARc activity was enhanced further in CHO-GPAT1 cells treated with the PPARc ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARc had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARc activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARc. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARc and that intermediates of de novo glycerolipid synthesis regulate gene expression.
Figure 2. FABP inhibitors reduce nociception in models of inflammatory and visceral pain. (A) Effects of SBFI26, SBFI50, SBFI60, and SBFI62 (20 mg/kg, i.p.) upon carrageenan-induced thermal hyperalgesia (left panel) and paw edema (right panel) in mice. *, p,0.05; **, p,0.01 versus carrageenan injected animals (black bar) (n = 6). (B) Effect of FABP inhibitors (20 mg/kg, i.p.) upon the first (left panel) and second phases (right panel) of formalin-induced nociception in mice. *, p,0.05 versus vehicle control (n = 6). (C) SBFI26 reduces acetic acid-induced writhing in mice. **, p,0.01 (n = 6). (D) Dose-response of SBFI26-mediated inhibition of acetic acid writhing in mice. **, p,0.01 (n = 6). (E) The antinociceptive effects of SBFI26 are reversed by the cannabinoid receptor 1 antagonist rimonabant (SR1, 3 mg/kg) and theperoxisomeproliferator-activatedreceptor alpha antagonist GW6471 (4 mg/kg). In contrast, the cannabinoid receptor 2 antagonist SR144518 (SR2, 3 mg/kg) and the opioid antagonist naloxone (2 mg/kg) were without effect. Rimonabant also reversed the antinociceptive effects ofthe FAAH inhibitor PF-3845 (blue bars). When administered alone, the antagonists did not modulate nociception (green bars). *, p,0.05; **, p,0.01 versus vehicle control. ##, p,0.01; ###, p,0.001 versus SBFI26 treated mice (n = 6–9).
RNA-induced silencing complex where they typically recognize and bind to sequences in the 39 untranslated regions, leading to suppression of translation and/or degradation of mRNA. There is accumulating evidence that miRNA play a critical role in immunity and inflammation [31–33]. For instance, distinct miRNA expression profiles have been found in Crohn’s disease and ulcerative colitis . By comparing miRNA profiles from C. difficile-infected and uninfected mice, we found that miR-146b, miR-1940, and miR-1298 were overexpressed in colons of C. difficile-infected mice. We focused our efforts on the miRNA-146 family (miRNA-146a/b) in this study since we validated its upregulation in the colon of C. difficile-infected mice by using RT- PCR. In addition, miRNA-146 is expressed in leukocytes and its function is clearly linked to innate immunity and inflammation [50,51]. miRNA-146 regulatory circuit improves TLR4 and cytokine signaling in response to microbial components and proinflammatory cytokines [52,53]. Moreover, it is involved in the regulation of T- and B-cell development [32,33], differentiation and function . To further understand the role of miRNA-146b during C. difficile infection, a list of mRNA potential targets for such miRNA was retrieved from miRBase . Notably, one ofthe co-activators facilitating the transcriptional activities oftheligand-activated PPARc, NCOA4, was a predicted target of miRNA-146b. Indeed, gene expression analyses using qRT-PCR demonstrated co-expression of miRNA-146b and NCOA4 in colons of C. difficile-infected mice and a negative correlation between expression of miR-146b and its target NCOA4 along with increasing doses of C. difficile, suggesting a potential inhibition of NCOA4 by miR-146b resulting in suppressed PPARc activity, as measured by suppressed expression of PPAR c-responsive genes (i.e., CD36 and Glut4) in C. difficile-infected mice. Thus, miRNAs become promising therapeutic targets once the functional con- sequences of miRNAs alteration are completely elucidated. Also, future studies should examine more direct therapeutic approaches to prevent overexpression of miRNA-146 during CDAD.
assays, the cells were removed by trypsinization and replated in 24 wells plate at density of 1,2610 5 cells/well. Cell transfections were performed using FuGENE 6 transfection reagent (Roche, Swiss) with 100 gg of plasmids containing wild-type PPARa,d ou c-LBD or PPARd-LBD mutants, DBD Gal-4, 50 gg of luciferase reporter plasmid and 1 gg of Renilla luciferase plasmid per well. Cells were treated with different concentrations of agonists of PPARa - GW7647, PPARd - GW0742 and PPARc - Roziglitazone, in triplicate 24 h after transfection and incubated for additional 24 h. Cell lysates were prepared and luciferase assay was performed using the Dual-Luciferase Report Assay system (Promega, Madison, WI), following manufacturer instructions. Light emission was measured by integration over 5 seconds of reaction in a Safire luminescent counter (Tecan, Tecan US, NC, USA). Firefly luciferase activity was normalized by the level of Renilla luciferase activity, as recommended by manufacturers Dual-Luciferase Report Assay system. Data were fitted using a sigmoidal dose-response function with corresponding EC50 determination according to GraphPad Prism software (version 5.0).
Fermented cottonseed meal (FCSM) is widely used in poultry diets in China. This study was conducted to investigate the effect of FCSM on lipid-related gene expression in broilers. Initially, 180 broiler chickens (21-days-old, equal number of males and females) were randomly divided into three groups, with six pens per group and 10 birds per pen. The chickens in the control group were fed a diet containing unfermented cottonseed meal, and those in the treatment groups were fed with diets including either CSM fermented by Candida tropicalis (Ct group) or CSM fermented by Candida tropicalis plus Saccharomyces cerevisae (Ct-Sc group) until 64 days old. The results revealed that, compared with the control group (p<0.05), the mRNA expression ofperoxisomeproliferator-activatedreceptor alpha (PPAR-α) and lipoprotein lipase (LPL) were upregulated in the livers of Ct-Sc males. The expression of PPAR-α was also upregulated in the livers of Ct females. The expression levels of acetyl CoA carboxylase (ACC) and LPL in the liver of males and the expression of PPAR-α in the liver of females were significantly different between the Ct and Ct-Sc groups (p<0.05). However, gene expressions of fatty acid synthase (FAS) and liver fatty acid-binding protein (L-FABP) in the liver were not altered when the broilers were fed FCSM-supplemented diets (p>0.05). Likewise, the expressions ofperoxisomeproliferator-activatedreceptor gamma (PPAR-γ) and LPL in the abdominal fat were not altered by the FCSM- supplemented diets (p>0.05). The results in this study indicate that CSM fermented by Candida tropicalis and Saccharomyces cerevisiae effectively regulated the genes involved in fatty acid β-oxidation and triglyceride hydrolysis in male broiler chickens. Furthermore, the effects ofthe FCSM-supplemented diets were significantly different between bird sexes and between yeast strains used in the fermentation process.
PPARα is a ligand-activated transcription factor that is one of three different PPAR subtypes: PPARα, PPARβ/δ, and PPARγ. The PPARs play important roles in nutrient homeostasis [11-13] and are localized in the nucleus. Although MECR was previous- ly reported as a binding protein of PPARα , interaction be- tween MECR and PPARα seems not to occur in mammalian cells due to their different subcellular localizations, mitochon- dria and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for function- al interaction between MECR and PPARα in the nuclei of cells. Here, we analyzed the expression pattern of MECR in sev- eral rat tissues and found a novel splice variant of MECR in which an additional exon was inserted between exon 1 and exon 2. The protein generated from this splicing variant has an N-terminal region that does not contain the mitochondrial tar- geting signal peptide and thus is not localized in mitochondria. Moreover, this MECR variant bound PPARα in the nucleus and enhanced PPARα transcriptional activity. Based on these results, we propose that this novel variant of MECR, cytosolic MECR (cMECR), plays a role in intracellular signal pathways as an interacting partner of PPARα.
The anti-tumor activity of PPARγ agonists on tumor cell lines and animal models have been well described [19, 35, 36]. PPAR ligands are known to induce cell cycle arrest [37, 38] as well as differentiation and apoptosis in different cell line models [19, 35], however the molecu- lar mechanism of their action as anticancer agents remains, so far, unclear. Although their effect on the lipid metabolism and glycemic control is well documented, thus, the PPARγ regu- latory pathway has been extensively investigated. Briefly, PPARγ binds to RXR (Retinoid X Receptor) and forms a heterodimeric complex which interacts with cognate sequences in pro- moter regions of target genes, by binding to specific DNA sequence elements termed PPRE (PeroxisomeProliferator Response Element) . In the absence of a ligand, PPARs/RXR het- erodimer remains inactive through its binding with several co-repressors [40–42], while in the presence of a ligand, for either PPARγ or RXR, the co-repressors dissociate and theligand can bind and activate several co-activators (Fig 7) . In this regard, it has been reported that simultaneous treatments with ligands, specific for both PPARγ and RXR, has a synergistic effect on the transactivation of reporter genes. Several identified co-activators and co-repres- sors of PPARγ have intrinsic histone-modifying activities such as HATs and HDACs, which are acting as co-activators and co-repressors of PPARγ, respectively (Fig 7) [40, 43].
upregulated if PPARc was directly overexpressed in THP-1 cells. Using lentiviral gene delivery, we overexpressed PPARc (PPARc THP-1) or a GFP-control (GFP THP-1) directly in the THP-1 cells. After one week of expansion, equal numbers of cells were plated into a tissue culture plate and left unactivated or activated with LPS or PAM3CSK4 for 24 hours, after which, non-adherent cells were aspirated, the wells were washed three times in phosphate buffered saline, and attached cells were imaged and quantified with an inverted microscope. In all conditions, PPARc THP-1 cells were more adherent than control THP-1 cells (Figure 6C). We also collected total mRNA from unactivated cultures to quantify levels of fibronectin and the fibronectin binding integrin, ITGA5 (Figure 6D). Compared to the control transduction, THP-1 cells that overexpressed PPARc had a significant increase in fibronectin levels. To further support the upregulation of these two molecules, we next investigated protein levels in transduced THP-1 cells. Cells were fixed and permeablized for immunostaining, followed by analysis via flow cytometry. GFP+ events were pre-gated, and the mean fluorescence intensity (MFI) of fibronectin or ITGA5 protein, minus unstained background values, were analyzed (Figure 6F). Both fibronectin and ITGA5 protein expression were significantly increased in THP-1 cells that overexpressed PPARc.
On the present day, almost all positions ofthe vitamin D structure have been modified in order to create new analogs. Most ofthe modifications are produced in the side chain since it is directly related to the biologic response and selectivity ofthe compounds. The integrity ofthe triene system is essential for the biological activity ofthe vitamin and therefore few analogs have alterations at this site. The CD bicycle is also usually left untouched, due to lack of information on the structure-activity correlation and to the difficulty in synthesizing such compounds.
attenuated neointima formation through glycogen synthase kinase-3b (GSK-3b) activation . Because GSK-3b is known as a negative regulator of platelet function ,_ENREF_38 this would be another mechanism of anti-thrombotic action ofthe PPAR-c agonist. Taken together, our finding suggests the possibility of its usefulness in the prevention of stent thrombosis. Furthermore, our data may have another clinical relevance; the considerable efficacy of short-term treatment (9 days) with rosiglitazone can give a chance to avoid potential adverse effects of this drug by ‘limiting the duration of treatment to periprocedural phase of stent implantation’. However, it is more plausible to evaluate the effects of rosiglitazone on TF and TFPI expressions in normal vessels without balloon injury as well as the effects of a prolonged exposure ofthe drug prior to balloon injury, since these models are more relevant to the clinical situation in which diabetic patients take the drug for a long time before having myocardial infarction. In this context, we also tried to analyze the effects of rosiglitazone on TF and TFPI expressions in corresponding contralateral uninjured carotid arteries. Al- though the effects ofthe PPAR-c agonist on TF and TFPI expressions in the uninjured arteries were not dramatic compared to those in the injured arteries, the drug could reverse paclitaxel- induced TF expression without reducing TFPI expression (Figure S8A-B). Accordingly, it can be speculated that rosiglitazone may have a protective, or, at least, no harmful effect on thrombogen- esis in the normal vessels as well as the vessels with stent implantation. Further experiments using diabetes animal model with long-term rosiglitazone administration would be the next step in order to validate this effect and to provide a biological answer to the concerns over cardiovascular safety of rosiglitazone. More importantly, thrombosis model are needed to confirm a hitherto unrecognized role of PPAR-c agonists: a potential treatment to reduce the risk of stent thrombosis. Moreover, future clinical researches for patients with drug-eluting stents implan- tation can build on these observations.
The in vivo experiments suggested that coordinated regulation of adiponectin, Sirt6, and AMPK may play a role in RGZ’s protective action. The up-regulation of Sirt6 and its related genes as well as the activation of LKB1 and AMPK by RGZ led us to hypothesize that RGZ may exert its positive effects by acting on Sirt6. We therefore performed RNAi-mediated gene silencing by transfecting AML12 cells with siRNA oligos targeting Sirt6. Subsets of cells were incubated with FFA for 48 h to induce hepatocyte steatosis, and then treated with FFA and/or RGZ for additional 24 h. FFA incubation for 72 h induced hepatocyte steatosis with a significant increase in TG and FFA (Figure 5). As expected, RGZ treatment significantly reduced hepatocyte lipid accumulation. However, Sirt6 knockdown significantly increased TG and FFA levels in FFA-treated hepatocytes and diminished the effects of RGZ on hepatocyte lipid accumulation (Figure 5). The mRNA and protein expression patterns of targets that were investigated were similar to those found in vivo (Figure 6). Sirt6 knockdown was confirmed by a significant repression of its mRNA and protein expression (Figures 6D, G, H). Gene expression of Adipoq, Ppara, and Pparg was up-regulated by RGZ and the up- regulation was not altered with Sirt6 knockdown, suggesting that these may act as upstream regulators of Sirt6 (Figures 6A–C). However, the RGZ-mediated up-regulation of Ppargc1a/PGC1-a and Foxo1 was abolished with Sirt6 knockdown, suggesting that Ppargc1a/PGC1-a and Foxo1 are downstream targets of Sirt6 in AML12 mouse hepatocytes (Figures 6E–H). Sirt6 knockdown has no effect on Sirt1 mRNA expression level (control siRNA, 1.0760.31; Sirt6 siRNA, 1.1160.21 relative fold change; p = 0.888).
on race, with black subjects having a significantly higher expression level compared to non-black subjects (0.0101 vs. 0.0125; p=0.03). However, there was a significantly lower level of PPARg mRNA expression in HCV-HIV-co-infected subjects compared to HCV-only subjects in both groups [non-black (p=0.001) (Figure 2B) and black subjects (p=0.03)] (Figure 2C). Other factors evaluated and found to have no association with PPARg gene expression include age, sex, BMI, HCV genotype, HCV RNA serum levels, DM, alcohol use, CD4 counts, serum HIV RNA levels and HAART therapy (Table 2). In multivariate linear regression analyses, only HIV-co-infection (p=0.001) and non-black race (p=0.04) were independently associated with lower PPARg expression (Table 2). The pathology findings demonstrated that more advanced fibrosis in liver biopsy was associated with lower hepatic PPARg mRNA expression among non-black, non-HIV subjects (METAVIR score F2/F3/F4 0.0102 vs. METAVIR score F0/F1 0.0126; p=0.04). There was no association between PPARg mRNA expression and steatosis in any group.
Microarray data showed that chitosan downregulated the expressions of apoB and ghrelin genes in the stomach. ApoB, a large amphipathic protein, is mainly expressed in the liver and is present on very-low density lipoproteins (VLDL), intermediate density lipoproteins, and low-density lipoproteins. ApoB is required for the formation of VLDL in the liver. Bindingof apoB to the microsomal transport protein results in the incorporation of lipids into the apoB molecule and leads to the formation of VLDL particles [30,41]. In clinical practice, apoB can be used as a marker to estimate the total number of atherogenic lipoprotein particles . Elevated apoB is a hallmark of several inherited disorders associated with atherosclerosis . However, patients with extremely low levels of apoB seem to be protected against cardiovascular diseases . Because apoB is an essential component of lipoprotein, the down-regulated expression of apoB gene by chitosan might contribute to the hypotriglyceridemic and hypocholesterolemic effects of chitosan. Ghrelin is a peptide hormone mainly produced by the stomach. Ghrelin is a potent stimulator of growth hormone secretion . Moreover, it is the only circulatory hormone that potently enhances the feeding and weight gain, increases the gastrointestinal mobility, and regulates Table 3. Expression levels of ghrelin, apoB, xpo4, pin1, penk1, and prdx2 genes by qPCR.
Autodock Ligand Docking Calculations with a partially flexible binding site selection were able to generate chemically reasonable protein ligand complex geometries for ATA-3 binding to the iGluR bindingdomain. The reliability ofligand docking results was validated by re-docking a series of seven ligands of known structure and obtaining excellent agreement ofthe top ranked placement to the known binding geometry. The single exception, the natural ligand glutamate for which only the second placement corresponded to the experimental binding mode, can be understood as well, since the glutamate ion is a small, highly polar molecule very unlike typical drug compounds for which docking tools are developed and may therefore represent a case were the autodock scoring function is unreliable. Even in this case, the correct binding mode is found, if not ranked perfectly. Using a variety of experimentally known receptor structures corresponding to different levels of LBD closure gives quite different docking results for ATA-3. No binding poses were suggested for the cis- configuration oftheligand. Only for one receptor X-ray structure (2P2A) we found a binding mode of trans-ATA-3 that resembles that ofthe similar compound 2-BnTetAMPA. For one additional, slightly more open receptor structure (1FTK), a second, unexpected binding pose was predicted for trans-ATA-3. This suggests that i) we have found a credible binding mode and a Figure 8. a) The distance between Gly451 and Ser651 after the back-isomerization to trans; b) The distance between E402 and T686 after back- isomerization.
(family Orthomyxoviridae; [5,20]), but also in toro- and coronaviruses, positive-stranded RNA viruses in the order Nidovirales [21,22]. From phylogenetic and comparative structural analyses it appears that toro- and coronaviruses acquired their HE proteins separately via horizontal gene transfer, with an (hemagglutinin-esterase- fusion) HEF-like protein as progenitor [22–25]. Like influenza C virus HEF, most nidovirus HEs bind to 9-O-acetylated (9-O-Ac) Sias and, correspondingly, display sialate-9-O-acetylesterase re- ceptor-destroying enzyme activity . Murine coronaviruses, however, occur in two closely related biotypes that differ in HE ligand/substrate preference. One of these -represented by mouse hepatitis virus (MHV) strain DVIM- displays the presumptive ancestral specificity and targets 9-O-Ac-Sias, while the other -represented by MHV strain S- appears to have evolved to use 4- O-Ac-Sias instead [6,25–27] (for supplementary introduction see Text S1 and Figure S1). Given the stereochemical differences between these Sia variants (Figure 1) and the essentially different requirements for ligand and substrate recognition by the respective HEs, the question arises how this major shift in receptor usage was achieved and what changes must have occurred in thereceptor- binding and O-acetylesterase domains to make this transition possible.
SmRTK1 is a membrane protein with an extracellular bindingdomain similar to several protein domains that share the Venus Flytap-VFT structure and the cytoplas- matic TK domain, which is similar to the insulin receptor (IR) catalytic domain. The SmRTK1 gene is expressed throughout all developmental stages. In males, it is pref- erably found in parenchyma cells. In females, an intense labelling was associated with ovocytes present in the ovary and in the ovary duct. SmRTK1 is believed to con- stitute an original GABA-activated RTK, which is involved in pheromone recognition, necessary for the development ofthe female ovaries (Vicogne et al. 2003). SmRTK1 was localised in sporocysts. The preferential localisation of SmRTK1 in sporocysts germinal cells and ovocytes could point to a role in schistosome growth and differentiation. SmIR-1 is a tyrosine kinase similar to the family mem- bers of IR. It has all the features of IR with a conserved ligand-bindingdomain. Immunohistochemical studies have shown that SmIR-1 is mainly expressed at the basal membrane level ofthe tegument in adult worms (Dissous et al. 2006, Khayath et al. unpublished results). It might play a role in glucose uptake regulation.