Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol

Rhoptries are secretory organelles involved in the virulence of the human pathogen Toxoplasma gondii. In the present study we have used HPLC and capillary GLC to isolate and quantify lipids from whole Toxoplasma cells and their purified rhoptries. This comparative lipidomic analysis revealed an enrichment of cholesterol, sphingomyelin and, most of all, saturated fatty acids in the rhoptries. These lipids are known, when present in membranes, to contribute to their rigidity and, interestingly, fluorescence anisotropy measurements confirmed that rhoptry-derived membranes have a lower fluidity than membranes from whole T. gondii cells. Moreover, although rhoptries were initially thought to be highly enriched in cholesterol, we demonstrated that cholesterol is present in lower proportions, and we have provided additional evidence towards a lack of involvement of rhoptry cholesterol in the process of host-cell invasion by the parasite. Indeed, depleting the cholesterol content of the parasites did not prevent the secretion of protein-containing rhoptry-derived vesicles and the parasites could still establish a structure called the moving junction, which is necessary for invasion. Instead, the crucial role of host cholesterol for invasion, which has already been demonstrated [Coppens and Joiner (2003) Mol. Biol. Cell 14, 3804-3820], might be explained by the need of a cholesterol-rich region of the host cell we could visualize at the point of contact with the attached parasite, in conditions where parasite motility was blocked.     

Mathematical model of nucleotide regulation on airway epithelia. Implications for airway homeostasis

In the airways, adenine nucleotides support a complex signaling network mediating host defenses. Released by the epithelium into the airway surface liquid (ASL) layer, they regulate mucus clearance through P2 (ATP) receptors, and following surface metabolism through P1 (adenosine; Ado) receptors. The complexity of ASL nucleotide regulation provides an ideal subject for biochemical network modeling. A mathematical model was developed to integrate nucleotide release, the ectoenzymes supporting the dephosphorylation of ATP into Ado, Ado deamination into inosine (Ino), and nucleoside uptake. The model also includes ecto-adenylate kinase activity and feed-forward inhibition of Ado production by ATP and ADP. The parameters were optimized by fitting the model to experimental data for the steady-state and transient concentration profiles generated by adding ATP to polarized primary cultures of human bronchial epithelial (HBE) cells. The model captures major aspects of ATP and Ado regulation, including their >4-fold increase in concentration induced by mechanical stress mimicking normal breathing. The model also confirmed the independence of steady-state nucleotide concentrations on the ASL volume, an important regulator of airway clearance. An interactive approach between simulations and assays revealed that feed-forward inhibition is mediated by selective inhibition of ecto-5'-nucleotidase. Importantly, the model identifies ecto-adenylate kinase as a key regulator of ASL ATP and proposes novel strategies for the treatment of airway diseases characterized by impaired nucleotide-mediated clearance. These new insights into the biochemical processes supporting ASL nucleotide regulation illustrate the potential of this mathematical model for fundamental and clinical research.     

Endothelial and vascular dysfunctions and insulin resistance in rats fed a high-fat, high-sucrose diet

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis.     

An olfactory receptor pseudogene whose function emerged in humans: a case study in the evolution of structure–function in GPCRs

Human olfactory receptor, hOR17-210, is identified as a pseudogene in the human genome. Experimental data has shown however, that the gene product of frame-shifted, cloned hOR17-210 cDNA was able to bind an odorant-binding protein and is narrowly tuned for excitation by cyclic ketones. Supported by experimental results, we used the bioinformatics methods of sequence analysis (genome-wide and pair-wise), computational protein modeling and docking, to show that functionality in this receptor is retained due to sequence-structure features not previously observed in mammalian ORs. This receptor does not possess the first two transmembrane helical domains (of seven typically seen in GPCRs). It however, possesses an additional TM that has not been observed in other human olfactory receptors. By incorporating these novel structural features, we created two putative models for this receptor. We also docked odor ligands that were experimentally shown to bind hOR17-210. We show how and why structural modifications of OR17-210 do not hinder this receptor's functionality. Our studies reveal that novel gene rearrangements that result in sequence and structural diversity may have a bearing on OR and GPCR function and evolution.     

Toxoplasma gondii autophagy‐related protein ATG9 is crucial for the survival of parasites in their host

Autophagy is a conserved, life‐promoting, catabolic process involved in the recycling of nonessential cellular components in response to stress. The parasite Toxoplasma gondii is an early‐diverging eukaryote in which part of the autophagy machinery is not exclusively involved in a catabolic process but instead has been repurposed for an original function in organelle inheritance during cell division. This function, depending essentially on protein TgATG8 and its membrane conjugation system, is crucial for parasite survival and prevented an in depth study of autophagy in the mutants generated so far in Toxoplasma. Thus, in order to decipher the primary function of canonical autophagy in the parasites, we generated a cell line deficient for TgATG9, a protein thought to be involved in the early steps of the autophagy process. Although the protein proved to be dispensable for the development of these obligate intracellular parasites in vitro, the absence of TgATG9 led to a reduced ability to sustain prolonged extracellular stress. Importantly, depletion of the protein significantly reduced parasites survival in macrophages and markedly attenuated their virulence in mice. Altogether, this shows TgATG9 is important for the fate of Toxoplasma in immune cells and contributes to the overall virulence of the parasite, possibly through an involvement in a canonical autophagy pathway.     

The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes.

Chlorophyll biosynthesis requires a metabolic dialog between the chloroplast envelope and thylakoids where biosynthetic activities are localized. Here, we report the first plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase (MgP(IX)MT) sequence identified in the Arabidopsis genome owing to its similarity with the Synechocystis sp. MgP(IX)MT gene. After expression in Escherichia coli, the recombinant Arabidopsis thaliana cDNA was shown to encode a protein having MgP(IX)MT activity. The full-length polypeptide exhibits a chloroplast transit peptide that is processed during import into the chloroplast. The mature protein contains two functional regions. The C-terminal part aligns with the Synechocystis full-length protein. The corresponding truncated region binds to Ado-met, as assayed by UV crosslinking, and is shown to harbor the MgP(IX)MT activity. Downstream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane. In A. thaliana and spinach, the MgP(IX)MT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids. The protein is active in each membrane and has the same apparent size corresponding to the processed mature protein. The protein is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane. The rationale for a dual localization of the protein in the chloroplast is discussed.     

Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes.

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.     

Impact of engagement of FcepsilonRI and CC chemokine receptor 1 on mast cell activation and motility

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.     

Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

Glial localization of interleukin-1α in invertebrate ganglia

1. Mytilus pedal ganglion contains a small population of glial cells that are immunopositive for interleukin-1 alpha. Positively stained fibers can also be seen in the neuropil of these sections. 2. The marine worm Nereis diversicolor also exhibits positive neural immunostaining for interleukin-1 alpha. 3. Both organisms contain hemocytes that contain immunoactivity for interleukin-1 alpha. The study suggests interleukin-1 alpha to be an ancient cytokine given its presence in organisms that evolved significantly earlier than mammals.