Regulation of a pentameric ligand-gated ion channel by a semiconserved cationic lipid-binding site

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure–function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABA_A receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABA_A receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.     

Immunostimulating effects of a sulfated galactan from Codium fragile

A pyruvylated sulfated galactan from Codium fragile is a highly ramified polysaccharide consisting of 3-linked, 3,6-linked, and non-reducing terminal d-galactose with pyruvate and sulfate groups; the glycan exerts anti-herpes simplex virus type 2 effects in vitro and in vivo. This particular polysaccharide was found to stimulate the production of nitric oxide by inducing iNOS at the mRNA and protein levels. In addition, the polysaccharide also induced several cytokine mRNA expressions such as IL-1β, IL-6, IL-10 and TNF-α. Therefore, it appears that the sulfated galactan might possess the immunostimulating effects via activation of macrophages.     

Plasmodium falciparum: the fungal metabolite gliotoxin inhibits proteasome proteolytic activity and exerts a plasmodicidal effect on P. falciparum

The in vitro antimalarial activity of the fungal metabolite gliotoxin (GTX) was evaluated, and its mechanism of action was studied. GTX showed plasmodicidal activity against both Plasmodium falciparum chloroquine-resistant strain K-1 and chloroquine-susceptible strain FCR-3. GTX cytotoxicity was significantly lower against a normal liver cell line (Chang Liver cells). The intracellular reduced glutathione level of parasitized and of normal red blood cells was not affected by GTX treatment. However, GTX decreased the chymotrypsin-like activity of parasite proteasomes in a time-dependent manner. The results of this study indicate that GTX possesses plasmodicidal activity and that this effect is due to inhibition of parasite proteasome activity, suggesting that GTX may constitute a useful antimalarial therapy.     

A lycoperdaceous fungus, Arachnion album (Agaricales, Arachniaceae), newly found in Japan

Arachnion album was collected in Osaka and Hyogo Prefectures, western Honshu, Japan. This is a new record of this genus for Japan. Macro- and microscopic features of the species are described and illustrated based on the Japanese specimens, and comparisons with some related taxa of A. album are given.     

Masking oligonucleotides improve sensitivity of mutation detection based on guanine quenching

Guanine quenching of a fluorescence-labeled DNA probe is a powerful tool for detecting a mutation in a targeted site of a DNA strand. However, a different guanine adjacent to a targeted site can interfere with detection of a point mutation, resulting in unsatisfactory sensitivity. In the current study, we developed a simple method to improve sensitivity of the guanine quenching method using a masking DNA oligonucleotide. The simple addition of a masking DNA oligonucleotide was found to mask the interference of a different guanine in a target oligonucleotide on fluorescence and to enhance difference in the quenching ratio between wild-type and mutant oligonucleotides. Based on this strategy, we succeeded in discriminating various mutations from the wild-type YMDD motif of the hepatitis B virus DNA polymerase gene using guanine quenching with a masking oligonucleotide.     

Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry

An oral sorbent AST-120 composed of spherical porous carbon particles has superior adsorption ability for certain small-molecular-weight organic compounds known to accumulate in patients with chronic renal failure (CRF). A metabolomic approach was applied to search for uremic toxins as possible indicators of the effect of AST-120. Serum metabolites in normal and CRF rats before and after administration of AST-120 for 3 days were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis. Further, serum and urine levels of the indicators were quantified by selected reaction monitoring of LC/ESI-MS/MS. Indoxyl sulfate was the first principal serum metabolite, which could differentiate CRF from both normal and AST-120-administered CRF rats, followed by hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate. CRF rats showed increased serum levels of indoxyl sulfate, hippuric acid, phenyl sulfate, 4-ethylphenyl sulfate and p-cresyl sulfate. Administration of AST-120 for 3 days to the CRF rats reduced the serum and urine levels of these metabolites. In conclusion, indoxyl sulfate is the best indicator of the effect of AST-120 in CRF rats. Hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate are suggested as the additional indicators. 4-Ethylphenyl sulfate is a newly identified uremic substance.     

NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells

We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated β-galactosidase activity and expression of p53, transforming growth factor (TGF)-β1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, β-galactosidase, TGF-β1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.     

Baicalein 5,6,7-trimethyl ether, a flavonoid derivative, stimulates fatty acid beta-oxidation in skin fibroblasts of X-linked adrenoleukodystrophy

The purpose of the present study is to identify bioactive compounds with potential for X-linked adrenoleukodystrophy (X-ALD) pharmacological therapy. Various plant natural products including flavonoids were tested for their ability to ameliorate the abnormality of very long chain fatty acid (VLCFA) metabolism in cultured skin-fibroblasts from X-ALD patients. Of the compounds tested, baicalein 5,6,7-trimethyl ether (baicalein-tri-Me) was found to significantly stimulate the VLCFA beta-oxidation activity. Furthermore, the incorporation of [1-(14)C]lignoceric acid into cholesteryl esters was markedly reduced towards the normal level and the VLCFA (C24:0 and C26:0) content was decreased. These results make baicalein-tri-Me a candidate for the therapeutic compound for X-ALD.