Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide

TLR2 in association with TLR1 or TLR6 plays an important role in the innate immune response by recognizing microbial lipoproteins and lipopeptides. Here we present the crystal structures of the human TLR1-TLR2-lipopeptide complex and of the mouse TLR2-lipopeptide complex. Binding of the tri-acylated lipopeptide, Pam(3)CSK(4), induced the formation of an "m" shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam(2)CSK(4), did not. The three lipid chains of Pam(3)CSK(4) mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR1-TLR2 heterodimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.     

Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells

A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.     

Characterization of an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle

Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60 degrees . A broad range of lipase substrates, from C4 to C18 rho-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was rho-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.     

Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit

The brains of Alzheimer's disease (AD) patients are characterized by large deposits of amyloid beta peptide (Abeta). Abeta is known to increase free radical production in nerve cells, leading to cell death that is characterized by lipid peroxidation, free radical formation, protein oxi-dation, and DNA/RNA oxidation. In this study, we selected an extract of Gardenia jasminoides by screening, and investigated its ameliorating effects on Abeta-induced oxidative stress using PC12 cells. The effects of the extract were evaluated using the 2,7 -dichlorofluorescein diacetate (DCF-DA) assay and the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. To find the active component, the ethanol extract was partitioned with hexane, chloroform, and ethyl acetate, respectively, and the active component was purified by silica-gel column chromatography and HPLC. The results suggested that Gardenia jasminoides extract can reduce the cytotoxicity of Abeta in PC 12 cells, possibly by reducing oxidative stress.     

R-type pyocin is required for competitive growth advantage between Pseudomonas aeruginosa strains

R-type pyocin is a bacteriophage tail-shaped bacteriocin produced by Pseudomonas aeruginosa, but its physiological roles are relatively unknown. Here we describe a role of R-type pyocin in the competitive growth advantages between P. aeruginosa strains. Partial purification and gene disruption revealed that the major killing activity from the culture supernatant of PA14 is attributed to R-type pyocin, neither F-type nor S-type pyocins. These findings may provide insight into the forces governing P. aeruginosa population dynamics to promote and maintain its biodiversity.     

Factors affecting redox potential and differential sensitivity of SoxR to redox‐active compounds

SoxR is a [2Fe‐2S]‐containing sensor‐regulator, which is activated through oxidation by redox‐active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth‐inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe‐2S] binding site were proposed as key determinants of sensitivity. ScSoxR‐like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR‐like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxR^R127L/P131V and ScSoxR^L126R/V130P were estimated to be ?192 ± 8 mV and ?273 ± 10 mV, respectively, approaching that of ScSoxR (?185 mV) and EcSoxR (?290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe‐2S], making it harder to get oxidized. This reveals a mechanism to modulate redox‐potential in [Fe‐S]‐containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology.     

The expression of the receptor for advanced glycation end-products (RAGE) in RA-FLS is induced by IL-17 via Act-1

Introduction

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-κB (NF-κB) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated.

Methods

RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production.

Results

RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1β (*P <0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-α in RA-FLS. The combined stimuli of both IL-17 and IL-1β significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1β alone (P <0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL-17.

Conclusions

RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL-1β. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment.     

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 1?N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.     

Influence of diet enriched with green tea on innate humoral and cellular immune response of kelp grouper (Epinephelus bruneus) to Vibrio carchariae infection

Effect of diet enriched with green tea at 0, 0.01, 0.1 or 1.0% levels on immune responses such as non-specific humoral (lysozyme, antiprotease and complement) and cellular (myeloperoxidase content, production of reactive oxygen, and nitrogen species) and disease resistance on week 1, 2 or 4 in kelp grouper Epinephelus bruneus challenged with Vibrio carchariae (2.47 × 10(8) CFU ml(-1)) was quantified. At all doses green tea supplementation significantly enhanced the serum lysozyme activity from weeks 1 to 4. On the other hand, after week 2 the serum hemolytic complement activity, leucocyte myeloperoxidase content and reactive nitrogen species protection significantly increased in groups fed with 0.01 and 0.1% green tea supplementation diets. The serum antiprotease activity significantly increased in group fed with at 1.0% green tea from week 1 to 4. However, all diets except at 0.01% level resulted in a significant decrease in reactive oxygen species protection during the experimental period. Challenged groups fed with green tea enriched diet at 0.01 and 0.1% level had a higher relative percent survival than with 1.0% diet on week 1, 2 or 4. The results suggest that dietary administration of green tea supplementation at a concentration of 0.01 and 0.1% level positively enhances the non-specific humoral and cellular immune responses and disease resistance of kelp grouper E. bruneus to V. carchariae.