Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-kappaB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys(30) and Cys(36) in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-kappaB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.     

Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400

SEA0400 is a potent and selective Na(+)/Ca(2+) exchanger (NCX) inhibitor. We evaluated the inhibitory effects of SEA0400 on Na(+)(i)-dependent (45)Ca(2+) uptake and whole-cell Na(+)/Ca(2+) exchange currents in NCX-transfected fibroblasts. SEA0400 preferentially inhibited (45)Ca(2+) uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2. SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to SEA0400 without affecting that to KB-R7943. We also found that Gly-833-to-Cys mutation (within the alpha-2 repeat) greatly reduced the inhibition by SEA0400, but unexpectedly the NCX1 chimera with an alpha-2 repeat from NCKX2 possessed normal drug sensitivity. In addition, exchangers with mutated exchanger inhibitory peptide regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had a markedly reduced sensitivity or hypersensitivity to SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of SEA0400 in a hypoxic injury model using porcine renal tubular cells. SEA0400 protected against hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na(+)-dependent inactivation are critical determinants for the inhibition by SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by SEA0400.     

Isolation and characteristics of methanosaeta in paddy field soils

The population of filamentous acetate-utilizing methanogens in paddy field soils was 2.0 x 10(4) MPN/g dry soil in the submerged condition. They were able to form colonies in a deep agar medium, but not in a roll tube. Filamentous acetate-utilizing methanogens isolated from Kanagi, Japan (strain K-5) and Tsukuba, Japan (strain T-3) were divided into two types based on length of filaments. One type, strain K-5, formed a short chain which was dispersed easily by weak shaking. The other type, strain T-3, formed a long chain, which formed cotton-like flocs and was not dispersed by weak shaking. They had sheaths composed of a pair of adjacent membranes on the outside of the cell membranes. The 16S rRNA gene similarities of strain T-3 and K-5 to Methanosaeta concilii strain Opfikon were 100% and 99.5% respectively. Filamentous acetate-utilizing methanogens were also isolated from paddy field soils in various other regions of Japan. Our results suggest that Methanosaeta is universal in paddy soils and that it plays an important role in methane production from acetate.     

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.     

Phenotypic and genetic characterization of the Atp7a(Mo-Tohm) mottled mouse: a new murine model of Menkes disease

Mottled Tohoku (Atp7a(Mo-Tohm) or Mo(Tohm)) is an X-linked mutation with mottled pigmentation in heterozygous (Mo(Tohm)/+) females and is embryonic lethal at E11 in hemizygous (Mo(Tohm)/Y) males. Copper levels were low in the brain and high in the intestine of Mo(Tohm) mice. Two congenic strains with ICR or C57BL/6 (B6) background were produced for genetic and phenotypic analyses and revealed that Mo(Tohm)/+ females with ICR background survived until adulthood, while most with B6 background died within 2 days after birth. The Mo(Tohm)/Y males with both backgrounds died at around E11. Massive hemorrhage was shown in the yolk sac cavity with irregular attachment between the mesoderm and the endothelial cells of blood vessels in the embryos at E10.5, suggesting that this irregular attachment causes embryonic lethality. The Mo(Tohm) mutant had a 1440-bp deletion between intron 22 and exon 23 of the Atp7a gene. Mo(Tohm)/Y males with the wild-type Atp7a cDNA transgene were rescued from embryonic lethality, confirming that the Mo(Tohm) mutant is caused by the defect in the Atp7a gene. This mutant mouse is the most severe model of human Menkes disease in mottled mice established to date and one of the useful models for understanding the gene function of Menkes disease.     

Genome-wide expression analysis of yeast response during exposure to 4°C

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10°C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4°C. Hierarchical cluster analysis showed that the gene expression profile following 4°C exposure from 6 to 48 h was different from that at continuous 4°C culture. Under 4°C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4°C. The induction of heat shock proteins and glutathione at 4°C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.     

Vasohibin prevents arterial neointimal formation through angiogenesis inhibition

Vasohibin is a VEGF-inducible angiogenesis inhibitor in vascular endothelium. Here we examined the presence of vasohibin in human arterial wall, and found it in endothelium of adventitial microvessels in atherosclerotic lesion. Adventitial angiogenesis is involved in the progression of neointimal formation. Even in the presence of endogenous angiogenesis inhibitors, pathological angiogenesis persists. However, the supplementation of exogenous angiogenesis inhibitors can prevent pathological angiogenesis. We evaluated the potential role of vasohibin in neointimal formation. Adenovirus-mediated human vasohibin gene transfer in mouse liver resulted in the release of vasohibin in plasma and exhibited anti-angiogenic effects at remote sites. This gene transfer inhibited adventitial angiogenesis, macrophage infiltration, and neointimal formation after cuff placement on mouse femoral artery. Vasohibin exhibited no direct effect on migration and proliferation of smooth muscle cells. Thus, vasohibin has an activity to prevent neointimal formation by inhibiting adventitial angiogenesis.