Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.     

The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.     

Comparative analysis by frontal affinity chromatography of oligosaccharide specificity of GlcNAc-binding lectins, Griffonia simplicifolia lectin-II (GSL-II) and Boletopsis leucomelas lectin (BLL)

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.     

Assembly of a mixture of isomeric BChl c from Chlorobium limicola as determined by intermolecular 13C-13C dipolar correlations: coexistence of dimer-based and pseudo-monomer-based stackings

A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.     

Central role of endogenous Toll-like receptor-2 activation in regulating inflammation, reactive oxygen species production, and subsequent neointimal formation after vascular injury

Background

It is now evident that inflammation after vascular injury has significant impact on the restenosis after revascularization procedures such as angioplasty, stenting, and bypass grafting. However, the mechanisms that regulate inflammation and repair after vascular injury are incompletely understood. Here, we report that vascular injury-mediated cytokine expression, reactive oxygen species (ROS) production, as well as subsequent neointimal formation requires Toll-like receptor-2 (TLR-2) mediated signaling pathway in vivo.

Methods and results

Vascular injury was induced by cuff-placement around the femoral artery in non-transgenic littermates (NLC) and TLR-2 knockout (TLR-2KO) mice. After cuff-placement in NLC mice, expression of TLR-2 was significantly increased in both smooth muscle medial layer and adventitia. Interestingly, we found that inflammatory genes expression such as tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 were markedly decreased in TLR-2KO mice compared with NLC mice. In addition, ROS production after vascular injury was attenuated in TLR-2KO mice compared with NLC mice. Since we observed the significant role of endogenous TLR-2 activation in regulating inflammatory responses and ROS production after vascular injury, we determined whether inhibition of endogenous TLR-2 activation can inhibit neointimal proliferation after vascular injury. Neointimal hyperplasia was markedly suppressed in TLR-2KO mice compared with WT mice at both 2 and 4 weeks after vascular injury.

Conclusions

These findings suggested that endogenous TLR-2 activation might play a central role in the regulation of vascular inflammation as well as subsequent neointimal formation in injured vessels.     

Relationship of dietary intake of fish and non-fish selenium to serum lipids in Japanese rural coastal community.

Several studies have suggested that dietary selenium deficiency may be associated with an increased risk of coronary heart disease (CHD). In the present study, 55 men and 71 women were selected from participants in a health examination in a rural coastal community in Japan. The mean dietary selenium intake calculated from the simple food frequency questionnaire (SFFQ) was 127.5 micrograms/day. Fish was the major source of dietary selenium and it contributed to 68.7% of the daily total. HDL cholesterol was higher in the middle selenium intake group and in the high selenium intake group than in the low selenium intake group in all subjects and for males, and a significant difference was found between the middle selenium intake group and the low selenium intake group. The atherogenic index was significantly higher in the low selenium intake group than in the middle selenium intake group and in the high selenium intake group in males. GPx activity, total cholesterol and triacylglycerols did not show any significant differences among the three different selenium intake groups. Dietary intake of non-fish Se had a positive correlation with HDL cholesterol, and an inverse correlation with the atherogenic index in all subjects and for females. On the other hand, dietary intake of fish-Se had no relationship with any serum lipids. Non-fish Se is an important factor in selenium status for the prevention of CHD.     

Possible molecular mechanisms of species recognition by barnacle larvae inferred from multi-specific sequencing analysis of proteinaceous settlement-inducing pheromone

Gregarious settlement is essential for reproduction and survival of many barnacles. A glycoprotein, settlement-inducing protein complex (SIPC) has been recognized as a signal for settlement and it is expressed in both conspecific adults and larvae. Although the settlement-inducing activities of SIPC are species-specific, the molecular-based mechanism by which larvae distinguish conspecific SIPC from the SIPC of other species is still unknown. Here, the complete primary structure of the SIPC of Megabalanus coccopoma, as well as the partial structure of the SIPCs of Balanus improvisus, Megabalanus rosa, and Elminius modestus are reported. These SIPCs contain highly variable regions that possibly modulate the affinity for the receptor, resulting in the species specificity of SIPC. In addition, the distribution patterns of potential N-glycosylation sites were seen to be different among the various species. Differences in such post-translational modifications may contribute to the species specificity of SIPC.