Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling

The ASTRO-H X-ray Observatory

The joint JAXA/NASA ASTRO-H mission is the sixth in a series of highly successful X-ray missions initiated by the Institute of Space and Astronautical Science (ISAS). ASTRO-H will investigate the physics of the high-energy universe via a suite of four instruments, covering a very wide energy range, from 0.3 keV to 600 keV. These instruments include a high-resolution, high-throughput spectrometer sensitive over 0.3-2 keV with high spectral resolution of Delta E < 7 eV, enabled by a micro-calorimeter array located in the focal plane of thin-foil X-ray optics; hard X-ray imaging spectrometers covering 5-80 keV, located in the focal plane of multilayer-coated, focusing hard X-ray mirrors; a wide-field imaging spectrometer sensitive over 0.4-12 keV, with an X-ray CCD camera in the focal plane of a soft X-ray telescope; and a non-focusing Compton-camera type soft gamma-ray detector, sensitive in the 40-600 keV band. The simultaneous broad bandpass, coupled with high spectral resolution, will enable the pursuit of a wide variety of important science themes.Comment: 22 pages, 17 figures, Proceedings of the SPIE Astronomical Instrumentation "Space Telescopes and Instrumentation 2012: Ultraviolet to Gamma Ray

Hitomi (ASTRO-H) X-ray Astronomy Satellite

The Hitomi (ASTRO-H) mission is the sixth Japanese x-ray astronomy satellite developed by a large international collaboration, including Japan, USA, Canada, and Europe. The mission aimed to provide the highest energy resolution ever achieved at E  >  2  keV, using a microcalorimeter instrument, and to cover a wide energy range spanning four decades in energy from soft x-rays to gamma rays. After a successful launch on February 17, 2016, the spacecraft lost its function on March 26, 2016, but the commissioning phase for about a month provided valuable information on the onboard instruments and the spacecraft system, including astrophysical results obtained from first light observations. The paper describes the Hitomi (ASTRO-H) mission, its capabilities, the initial operation, and the instruments/spacecraft performances confirmed during the commissioning operations for about a month

The diacylated lipopeptide FSL-1 induces TLR2-mediated Th2 responses

The diacylated lipopeptide FSL-1 enhanced the generation of IgG antibodies in TLR2+/+ mice, but not in TLR2-/- mice, when administered together with hen egg lysozyme as an antigen. Escherichia coli lipopolysaccharide enhanced the generation of antigen-specific antibodies in both TLR2-/- and TLR2+/+ mice. In TLR2+/+ mice, the enhancement level by FSL-1 was similar to that by lipopolysaccharide. Analysis of the IgG antibodies subclass demonstrated that level of Th2-type IgG1 antibodies was higher than that of Th1-type IgG2a antibodies. Both FSL-1 and lipopolysaccharide induced production of IL-10 and IL-6 by splenocytes from TLR2+/+ mice. Lipopolysaccharide also induced production of these cytokines by splenocytes from TLR2-/- mice, but FSL-1 did not. Neither FSL-1 nor lipopolysaccharide induced IL-12p70 production by splenocytes from both types of mice. FSL-1 upregulated B7.2 expression in B220+ cells from TLR2+/+ mice but not those from TLR2-/- mice, whereas lipopolysaccharide upregulated B7.2 expression in B220+ cells from both types of mice. FSL-1 and, to a lesser extent, lipopolysaccharide activated mitogen-activated protein kinases in splenocytes. FSL-1 and, to a lesser extent,lipopolysaccharide induced the expression of c-Fos, which is known to be involved in Th2-type responses, in splenocytes. Thus, this study demonstrated that FSL-1 possessed TLR2-mediated Th2-type responses in vivo

Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2

Details of roles of carbohydrates attached to Toll-like receptors (TLRs) in the recognition of pathogen-associated molecular patterns and in the formation of the functional receptor complex still remain unknown. This study was designed to determine whether the glycans linked at Asn114, Asn199, Asn414 and Asn442 residues of TLR2 ectodomain were involved in the recognition of diacylated lipopeptide and lipoprotein. Single and multiple mutants were transfected into human embryonic kidney (HEK) 293 cells together with a NF-κB luciferase reporter plasmid. All of these mutants were expressed on the surface. SDS-PAGE of the transfectants demonstrated that these mutants migrated lower than wild-type TLR2 and their molecular masses decreased as the number of mutated Asn residues increased. TLR2N114A, TLR2N199A and TLR2N414A as well as wild-type TLR2 induced NF-κB activation when stimulated with these ligands, whereas TLR2N442A failed to induce NF-κB activation. All of triple and quadruple mutants failed to induce NF-κB activation, but were associated with both wild-type TLR2 and TLR6 in the transfectants. TLR2N114A,N199A, TLR2N114A,N414A and, to a lesser extent, TLR2N114A,N442A, in which two N-linked glycans are speculated to be exposed to the concave surface of TLR2 solenoid, not only induce NF-κB activation but also are associated with wild-type TLR2 and TLR6. These results suggest that the glycan at Asn442 and at least two N-linked glycans speculated to be exposed to the concave surface of TLR2 solenoid are involved in the recognition of ligands by TLR2 and/or in formation or maturation of a functional TLR2 receptor complex

Associations of Plasma Concentration Profiles of Dapagliflozin, a Selective Inhibitor of Sodium&ndash;Glucose Co-Transporter Type 2, with Its Effects in Japanese Patients with Type 2 Diabetes Mellitus

This study was conducted to evaluate the long-term plasma concentration profiles of dapagliflozin and its effects on the glycated hemoglobin (HbA1c) level, body weight, and estimated glomerular filtration rate (eGFR) in 72 Japanese outpatients with type 2 diabetes mellitus (T2DM) receiving metformin and a dipeptidyl peptidase-4 inhibitor. At baseline, HbA1c level, body weight, and eGFR were 6.9 &plusmn; 0.6%, 77.9 &plusmn; 13.5 kg, and 78.8 &plusmn; 20.7 mL/min/1.73 m2, respectively. A once-daily oral dose of 5 mg dapagliflozin was administered, and its trough plasma concentrations were evaluated at 1, 3, 6, 9, and 12 months. In this study, the patients with stable dapagliflozin concentrations were defined, based on a well-organized clinical trial, as those with average plasma concentrations of 2&ndash;5 ng/mL with a coefficient of variation &lt;30%; these values were achieved if patients complied with their once-daily dosage. Multivariate analysis showed a significant decrease in the HbA1c levels among patients with stable concentrations (&minus;0.6 &plusmn; 0.4%, p &lt; 0.01), which was greater than the mean change among all 72 patients (&minus;0.2 &plusmn; 0.5%, p &lt; 0.01). The patients&rsquo; mean body weight also decreased (&minus;2.3 &plusmn; 4.0 kg, p = 0.060). Average plasma concentrations ranged from 1.6 to 11.8 ng/mL; however, multivariate analysis indicated it was unrelated to the HbA1c-lowering effect. In conclusion, the long-term stability of plasma dapagliflozin concentration was important in lowering HbA1c level, and a once-daily oral dose of 5 mg was sufficient in achieving this effect

TLR2 signals triggered by mycoplasmal lipoprotein/lipopeptide induce K+ efflux to activate the NLRP3 inflammasome

The proinflammatory cytokine interleukin (IL)- 1β plays a crucial role in controlling bacterial infections and is produced after the processing of pro-IL- 1β by caspase-1, which is activated by the inflammasome. Mycoplasmal membrane lipoprotein and lipopeptide, which are typical Toll-like receptor 2 (TLR2) ligands, activate the NLRP3 inflammasome to produce IL-1β in macrophages, although the molecular mechanism behind this remains unclear. Here, we found that lipoproteins from Mycoplasma salivarium (MsLP) and M. pneumoniae (MpLP) and an M. salivarium-derived lipopeptide (FSL-1) exhibited IL-1β-inducing activity toward bone marrow-derived macrophages from C57BL/6 mice (TLR2+/+ BMMs), whereas the activity toward BMMs from TLR2-deficient mice (TLR2－/－ BMMs) was markedly reduced. Microarray analysis suggested that FSL-1 upregulates the potassium voltage-gated channel, subfamily F, member 1 (Kcnf1), which is involved in K+ efflux as one of the NLRP3 inflammasome activators, in a TLR2-dependent manner. Moreover, we found that a high extracellular concentration of K+, which blocks K+ efflux, downregulated the release of IL-1β. Thus, this study is the first to suggest that TLR2-mediated signals triggered by mycoplasmal lipoproteins/lipopeptide upregulate potassium channels to promote K+ efflux, by which the NLRP3 inflammasome is activated

Naringenin suppresses Toll-like receptor 2-mediated inflammatory responses through inhibition of receptor clustering on lipid rafts

Toll-like receptors (TLRs) are important innate immune receptors that sometimes cause excessive inflammatory responses and a perpetuated inflammatory loop that can be involved in inflammatory and autoimmune diseases. TLR2 recognizes bacterial lipoproteins in association with TLR1 or TLR6, and triggers inflammatory responses through activation of the transcription factor NF-κB. Naringenin, a type of citrus flavonoid, has been shown to possess anti-inflammatory properties, but its detailed action against TLR2 remains to be fully elucidated. The present study was designed to determine whether naringenin affects the inflammatory responses triggered by TLR2. Naringenin inhibited proinflammatory cytokine production and attenuated NF-κB activation in cells stimulated with a synthetic triacylated-type lipopeptide known as a TLR2/TLR1 ligand, as well as a synthetic diacylated-type lipopeptide known as a TLR2/TLR6 ligand. Moreover, a similar inhibitory effect was observed in cells stimulated with a crude lipophilic fraction extracted from Staphylococcus aureus cell walls and in cells stimulated with S. aureus cells. Furthermore, we showed that such an effect is caused by inhibition of TLR2 clustering in lipid rafts on the cell membrane. These results suggest that naringenin suppresses the inflammatory responses induced by TLR2 signal transduction. Our findings indicate a novel anti-inflammatory property of naringenin, mediated through the regulation of cell surface TLR2 functioning