Mariner-like elements in Rhynchosciara americana (Sciaridae) genome: molecular and cytological aspects

Two mariner-like elements, Ramar1 and Ramar2, are described in the genome of Rhynchosciara americana, whose nucleotide consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1 contains several conserved amino acid blocks which were identified, including a specific D,D(34)D signature motif. Ramar2 is a defective mariner-like element, which contains a deletion overlapping in most of the internal region of the transposase ORF while its extremities remain intact. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 phylogenetically present high identity to mariner-like elements of mauritiana subfamily. Southern blot analysis indicated that Ramar1 is widely represented in the genome of Rhynchosciara americana. In situ hybridizations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A.

Expression of TLR-4 and -2 in peripheral mononuclear cells in renal transplant patients with TLR-4 gene polymorphism

Introduction: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome. Objective: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism. Methods: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-alpha after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry. Results: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-alpha secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01). Conclusion: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins. (C) 2010 Elsevier B.V. All rights reserved.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

4-Fluoro-2-methoxyphenol, an apocynin analog with enhanced inhibitory effect on leukocyte oxidant production and phagocytosis

Apocynin, a methoxy-substituted catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the roots of Picrorhiza kurroa, has been extensively used as a non-toxic inhibitor of the multienzymatic complex NADPH oxidase. We discovered that the analogous methoxy-substituted catechol, 4-Fluoro-2-methoxyphenol (F-apocynin), in which the acetyl group present in apocynin was changed to a fluorine atom, was significantly more potent as an inhibitor of NADPH oxidase activity, myeloperoxidase (MPO) chlorinating activity and phagocytosis of microorganisms by neutrophils; it was also as potent as apocynin in inhibiting tumor necrosis factor-alpha (TNF alpha) release by peripheral blood mononuclear cells. We attribute the increased potency of F-apocynin to its increased lipophilicity, which could facilitate the passage of the drug through the cell membrane. The inhibition of MPO chlorination activity, phagocytosis and TNF alpha release shows that apocynin and F-apocynin actions are not restricted to reactive oxygen species inhibition, but further studies are needed to clarify if these mechanisms are related. Like apocynin, F-apocynin did not show cell toxicity, and is a strong candidate for use in the treatment of inflammatory diseases. (C) 2011 Elsevier B.V. All rights reserved.

TLR4/MYD88-dependent, LPS-induced synthesis of PGE(2) by macrophages or dendritic cells prevents anti-CD3-mediated CD95L upregulation in T cells

Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E-2(PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE2 in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.

Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis

Background Epidemiological and experimental data suggest that bacteria] lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll-like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro-Type 1 T helper cells (Th 1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways, we used TLR4-, MyD88-, TRIF-, or IL-12/IFN-gamma-deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co-adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co-adsorbed onto alum impaired in dose-dependent manner OVA-induced Th2-mediated allergic responses such as airway eosinophilia, type-2 cytokines secretion, airway hyper-reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1 -affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL-12/IFN-gamma axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll-like receptor 4 agonists co-adsorbed with allergen onto alum down-modulate allergic lung disease and prevent the development of polarized T cell-mediated airway inflammation.

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of similar to 1.7 kb located in 2 chromosomal bands of 600-720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria

Background. Malaria is one of the most significant infectious diseases in the world and is responsible for a large proportion of infant deaths. Toll-like receptors (TLRs), key components of innate immunity, are central to countering infection. Variants in the TLR-signaling pathway are associated with susceptibility to infectious diseases. Methods. We genotyped single nucleotide polymorphisms ( SNPs) of the genes associated with the TLR-signaling pathway in patients with mild malaria and individuals with asymptomatic Plasmodium infections by means of polymerase chain reaction. Results. Genotype distributions for the TLR-1 I602S differed significantly between patients with mild malaria and persons with asymptomatic infection. The TLR-1 602S allele was associated with an odds ratio ( OR) of 2.2 ( P = .003; P(corrected) = .015) for malaria among patients with mild malaria due to any Plasmodium species and 2.1 ( P = .015; P(corrected) = .75) among patients with mild malaria due to Plasmodium falciparum only. The TLR-6 S249P SNP showed an excess of homozygotes for the TLR-6 249P allele in asymptomatic persons, compared with patients with mild malaria due to any Plasmodium species (OR 2.1; 95% confidence interval [CI], 1.1-4.2; P = .01; P(corrected) = .05), suggesting that the TLR-6 249S allele may be a risk factor for malaria ( OR, 2.0; 95% CI, 1.1-3.7; P = 0.01; P(corrected) = .05). The TLR-9-1486C allele showed a strong association with high parasitemia ( P < .001). Conclusions. Our findings indicate that the TLR-1 and TLR- 6 variants are significantly associated with mild malaria, whereas the TLR-9-1486C/T variants are associated with high parasitemia. These discoveries may bring additional understanding to the pathogenesis of malaria.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide`s primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Immobilization of glucose oxidase enzyme (GOD) in large pore ordered mesoporous cage-like FDU-1 silica

Large pore ordered mesoporous silica FDU-1 with three-dimensional (3D) face-centered cubic, Fm3m arrangement of rnesopores, was synthesized under strong acid media using B-50-6600 poly(ethylene oxide)-poly(butylene oxide)-poly(ethylene oxide) triblock copolymer (EO(39)BO(47)EO(39)), tetraethyl orthosilicate (TEOS) and trimethyl-benzene (TMB). Large pore FDU-1 silica was obtained by using the following gel composition 1TEOS:0.00735B50-6600:0.00735TMB:6HCl:155H(2)O. The pristine material exhibited a BET specific surface area of 684 m(2) g(-1), total pore volume of 0.89 cm(3) g(-1), external surface area of 49 m(2) g(-1) and microporous volume of 0.09 cm(3) g(-1). The enzyme activity was determined by the Flow Injection Analysis-Chemiluminescence (FIA-CL) method. For GOD immobilized on the FDU-1 silica, GOD supernatant and GOD solution, the FIA-CL results were 9.0, 18.6 and 34.0 U, respectively. The value obtained for the activity of the GOD solution with FIA-CL method is in agreement with the 35 U, obtained by spectrophotometry. (C) 2011 Elsevier B.V. All rights reserved.

Synthesis and structure of cage-like mesoporous silica using different precursors

In this work the synthesis of cubic, FDU-1 type, ordered mesoporous silica (OMS) was developed from two types of silicon source, tetraethyl orthosilicate (TEOS) and a less expensive compound, sodium silicate (Na(2)Si(3)O(7)), in the presence of a new triblock copolymer template Vorasurf 504 (EO(38)BO(46)EO(38)). For both silicon precursors the synthesis temperature was evaluated. For TEOS the effect of polymer dissolution in methanol and the acid solution (HCl and HBr) on the material structure was analyzed. For Na(2)Si(3)O(7) the influence of the polymer mass and the hydrothermal treatment time were the explored experimental parameters. The samples were examined by Small Angle X-ray Scattering (SAXS) and Nitrogen Sorption. For both precursors the decrease on the synthesis temperature from ambient, -25 degrees C, to -15 degrees C improved the ordered porous structure. For TEOS, the SAXS results showed that there is an optimum amount of hydrophobic methanol that contributed to dissolve the polymer but did not provoke structural disorder. The less electronegative Br-ions, when compared to Cl-, induced a more ordered porous structure, higher surface areas and larger lattice parameters. For Na(2)Si(3)O(7) the increase on the hydrothermal treatment time as well as the use of an optimized amount of polymer promoted a better ordered porous structure. (C) 2011 Elsevier B.V. All rights reserved.

X-ray photoelectron spectroscopy analysis of zirconium nitride-like films prepared on Si(100) substrates by ion beam assisted deposition

Thin zirconium nitride films were prepared on Si(l 00) substrates at room temperature by ion beam assisted deposition with a 2 keV nitrogen ion beam. Arrival rate ratios ARR(N/Zr) used were 0.19, 0.39, 0.92, and 1.86. The chemical composition and bonding structure of the films were analyzed with X-ray photoelectron spectroscopy (XPS). Deconvolution results for Zr 3d, Zr 3p(3/2), N 1s, O 1s, and C 1s XPS spectra indicated self-consistently the presence of metal Zr-0, nitride ZrN, oxide ZrO2, oxymnide Zr2N2O, and carbide ZrC phases, and the amounts of these compounds were influenced by ARR(N/Zr). The chemical composition ratio N/Zr in the film increased with increasing ARR(N/Zr) until ARR(N/Zr) reached 0.92, reflecting the high reactivity of nitrogen in the ion beam, and stayed almost constant for ARR(N/Zr) >= 1, the excess nitrogen being rejected from the growing film. A considerable incorporation of contaminant oxygen and carbon into the depositing film was attributed to the getter effect of zirconium. (C) 2007 Elsevier B.V. All rights reserved.

Magnetic hysteresis loop shift in NiFe(2)O(4) nanocrystalline powder with large grain boundary fraction

Magnetic properties of nanocrystalline NiFe(2)O(4) spinel mechanically processed for 350 h have been studied using temperature dependent from both zero-field and in-field (57)Fe Mossbauer spectrometry and magnetization measurements. The hyperfine structure allows us to distinguish two main magnetic contributions: one attributed to the crystalline grain core, which has magnetic properties similar to the NiFe(2)O(4) spinel-like structure (n-NiFe(2)O(4)) and the other one due to the disordered grain boundary region, which presents topological and chemical disorder features(d-NiFe(2)O(4)). Mossbauer spectrometry determines a large fraction for the d-NiFe(2)O(4) region(62% of total area) and also suggests a speromagnet-like structure for it. Under applied magnetic field, the n-NiFe(2)O(4) spins are canted with angle dependent on the applied field magnitude. Mossbauer data also show that even under 120 kOe no magnetic saturation is observed for the two magnetic phases. In addition, the hysteresis loops, recorded for scan field of 50 kOe, are shifted in both field and magnetization axes, for temperatures below about 50 K. The hysteresis loop shifts may be due to two main contributions: the exchange bias field at the d-NiFe(2)O(4)/n-NiFe(2)O(4) interfaces and the minor loop effect caused by a high magnetic anisotropy of the d-NiFe(2)O(4) phase. It has also been shown that the spin configuration of the spin-glass like phase is modified by the consecutive field cycles, consequently the n-NiFe(2)O(4)/d-NiFe(2)O(4) magnetic interaction is also affected in this process. (C) 2010 Elsevier B.V. All rights reserved.