Role of roots in differences in seed cadmium concentration among soybean cultivars—proof by grafting experiment

Soybean cultivars show significant differences in cadmium (Cd) concentrations in seeds, due primarily to genetics, not environmental factors. We previously suggested that low-Cd cultivars accumulate Cd in their roots and thus prevent its translocation to the rest of the plant. Through grafting experiments, we drew the following conclusions about Cd absorption and translocation: (1) The amount of Cd accumulated in shoots is determined by the Cd accumulation capacity of roots: cultivars with a small capacity to accumulate Cd in roots translocate more Cd and accumulate it in shoots; (2) The Cd concentration in shoots is determined by the Cd accumulation capacity of roots and the shoot productive ability of the scion cultivar; (3) The Cd tolerance of shoots differs among cultivars. Enrei, with a high-Cd accumulation capacity of roots, had a low Cd tolerance of shoots compared with Suzuyutaka and Hatayutaka, with a low Cd accumulation capacity of roots; (4) Cultivars differ in their distribution of Cd to seed; (5) These results show that seed Cd concentration is influenced by the differences among cultivars in ease of translocation of Cd to seed and in Cd accumulation capacity of roots.     

Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells

The endothelial cell-specific granule Weibel-Palade body releases vasoactive substances capable of modulating vascular inflammation. Although innate recognition of pathogens by Toll-like receptors (TLRs) is thought to play a crucial role in promotion of inflammatory responses, the molecular basis for early-phase responses of endothelial cells to bacterial pathogens has not fully been understood. We here report that human aortic endothelial cells respond to bacterial lipoteichoic acid (LTA) and synthetic bacterial lipopeptides, but not lipopolysaccharide or peptidoglycan, to induce Weibel-Palade body exocytosis, accompanied by release or externalization of the storage components von Willebrand factor and P-selectin. LTA could activate rapid Weibel-Palade body exocytosis through a TLR2- and MyD88-dependent mechanism without de novo protein synthesis. This process was at least mediated through MyD88-dependent phosphorylation and activation of phospholipase Cgamma. Moreover, LTA activated interleukin-1 receptor-associated kinase-1-dependent delayed exocytosis with de novo protein synthesis and phospholipase Cgamma-dependent activation of the NF-kappaB pathway. Increased TLR2 expression by transfection or interferon-gamma treatment increased TLR2-mediated Weibel-Palade body exocytosis, whereas reduced TLR2 expression under laminar flow decreased the response. Thus, we propose a novel role for TLR2 in induction of a primary proinflammatory event in aortic endothelial cells through Weibel-Palade body exocytosis, which may be an important step for linking innate recognition of bacterial pathogens to vascular inflammation.     

Synergistic contribution of CD14 and HLA loci in the susceptibility to Buerger disease

Buerger disease (BD) is an occulusive vascular disease of unknown etiology. Although cigarette smoking is a well-known risk factor of BD, genetic factors may also play a role in the etiology. Because chronic bacterial infection such as oral periodontitis is suggested to be involved in the pathogenesis of BD, gene polymorphisms involved in the infectious immunity might be associated with BD as the genetic factor(s). We have previously reported that HLA-DRB1*1501 and B54 was associated with BD in Japanese. In this study, polymorphisms in HLA-DPB1, DRB1 and B were analyzed in 131 Japanese BD patients and 227 healthy controls. In addition, we investigated a functional promoter polymorphism, −260 C > T, of CD14 that is a main receptor of bacterial lipopolysaccharide. It was found that the frequencies of CD14 TT genotype [37.4 vs. 24.2%, P = 0.008 OR = 1.87, 95% confidence interval (CI); 1.18, 2.97], DRB1*1501 (34.4 vs. 13.2%, P _c = 4.4 × 10⁻⁵, OR = 3.44, 95%CI; 2.06, 5.73) and DPB1*0501 (79.4 vs. 55.1%, P _c = 4.7 × 10⁻⁵, OR = 3.14, 95%CI; 1.93, 5.11) were significantly higher in the patients than in the controls, demonstrating that at least three genetic markers were associated with BD. Stratification analyses of these associated markers suggested synergistic roles of the genetic factors. Odds ratios ranged from 4.72 to 12.57 in individuals carrying any two of these three markers. These findings suggested that the susceptibility to BD was in part controlled by genes involved in the innate and adaptive immunity.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

A Genome-wide Approach to Identify the Genes Involved in Biofilm Formation in E. coli

Biofilm forming cells are distinctive from the well-investigatedplanktonic cells and exhibit a different type of gene expression.Several new Escherichia coli genes related to biofilm formationhave recently been identified through genomic approaches suchas DNA microarray analysis. However, many others involved inthis process might have escaped detection due to poor expression,regulatory mechanism, or genetic backgrounds. Here, we screeneda collection of single-gene deletion mutants of E. coli named‘Keio collection’ to identify genes required forbiofilm formation. Of the 3985 mutants of non-essential genesin the collection thus examined, 110 showed a reduction in biofilmformation nine of which have not been well characterized yet.Systematic and quantitative analysis revealed the involvementof genes of various functions and reinforced the importancein biofilm formation of the genes for cell surface structuresand cell membrane. Characterization of the nine mutants of function-unknowngenes indicated that some of them, such as yfgA that geneticallyinteracts with a periplasmic chaperone gene surA together withyciB and yciM, might be required for the integrity of outermembrane.     

Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.     

Improvement of bone strength and dermal thickness due to dietary edible bird's nest extract in ovariectomized rats

Oral administration of edible bird's nest extract (EBNE) improved bone strength and calcium concentration in the femur of ovariectomized rats. Dermal thickness was also increased by EBNE supplementation, whereas EBNE administration did not affect the serum estradiol concentration. These results suggest that EBNE is effective for the improvement of bone loss and skin aging in postmenopause all women.     

JAK2 is an important signal transducer in IL-33-induced NF-κB activation

IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-κB and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced IκBα degradation and NF-κB activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce IκBα degradation and NF-κB activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L. In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-κB in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-κB activation.