Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

N(2)O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N(2)O uptake and conversion of (15)N-N(2)O into (15)N-N(2). Free-living cells of USDA110 showed N(2)O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N(2)O reductase activity. When detached soybean nodules formed with USDA110 were fed with (15)N-N(2)O, they rapidly emitted (15)N-N(2) outside the nodules at a ratio of 98.5% of (15)N-N(2)O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N(2)O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N(2)O (0.34 ppm). These results indicate that the conversion of N(2)O to N(2) depends exclusively on the respiratory N(2)O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N(2)O.     

插入到细胞膜上的葡萄糖转运子4可不暴露其被光化学法标记的活性位点

胰岛素刺激骨胳肌产生磷脂酰肌醇3, 4, 5三磷酸(PI(3,4,5)P₃), 它是促进葡萄糖转运子4(GLUT4)与细胞膜融合的必要条件. 向肌肉细胞内导入PI(3,4,5)P₃可以模拟胰岛素刺激GLUT4与细胞膜融合的作用, 但不足以增加细胞摄取葡萄糖的量. 本研究目的是探讨PI(3,4,5)P₃与胰岛素作用不同的机制. 在骨骼肌细胞株(L6-GLUT4myc)中, 应用免疫反应方法检测细胞膜片上与特异性抗体反应的GLUT4的胞浆区羧基末端表位和胞外区myc表位的可用性; 使用不能渗透到细胞内的甘露糖-生物素衍生物Bio-LC-ATB-BMPA, 结合亲和光化学标记法检测GLUT4胞外区的活性位点. 相对于基础组, 100 nmol/L胰岛素和10 mmol/L PI(3,4,5)P₃分别使与myc结合的抗体量增加1.64倍和1.58倍. 胰岛素还使细胞膜上GLUT4的光化学标记量和细胞膜片上与羧基末端表位结合的抗体量分别增加了2.47倍和2.04倍, 而PI(3,4,5)P₃则无此作用. 在胰岛素作用下, 细胞膜片上与羧基末端表位结合的抗体量大于与myc表位结合的抗体量(分别为2.04和1.64倍). 结果表明: (i) 尽管PI(3,4,5)P₃能使GLUT4与细胞膜融合, 但不能使GLUT4胞外区的活性位点暴露; (ii) GLUT4胞外区活性位点的可用性与胞浆区羧基末端的可用性相关; (iii) 除了能刺激GLUT4与细胞膜融合, 胰岛素还使封闭GLUT4羧基末端的蛋白脱离. 推论胞浆内某种蛋白封闭羧基末端, 同样阻止甘露糖-生物素衍生物对GLUT4活性位点的标记, 并可能妨碍GLUT4转运葡萄糖.     

Comparison of the evolutionary distances among syngens and sibling species of Paramecium

The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.     

Synthesis of Pseudomonas quorum-sensing autoinducer analogs and structural entities required for induction of apoptosis in macrophages

The synthesis of the analogs of N-3-oxododecanoyl-L-homoserine lactone (1) and their structure-activity relationship for the apoptotic induction in macrophages, P388D1 cells, are described. It was revealed that the position of the oxo group in the acyl side chain in addition to the presence of the L-homoserine lactone unit is crucial for the apoptosis-inducing activity. Furthermore, the long acyl side chains with hydrophobic distal ends are preferable for the activity.     

Odorless benzenethiols in synthesis of thioglycosides and its application for glycosylation reactions

p-Octyloxybenzenethiol (2) was synthesized as a new odorless benzenethiol. Moreover, preparation of thioglycosides using 2 and their application for glycosylation reactions were attempted. As a result, it was found that the thioglycosides were as excellent glycosyl donors as 4-dodecylphenyl 1-thio-glycosides, which were previously reported by our group, and more useful than the previous donors in terms of fine chemistry in glycosylation reaction activated with silver triflate and N-iodosuccinimide (NIS). In addition, this method was applicable to the sialylation with NIS and triflic acid. All procedures from the preparation of thioglycosides to the glycosylation reaction could be attained completely under conditions where no malodor was generated.     

Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration. We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras-mediated phosphatydylinositol 3-kinase activation, and beta(1) integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras-specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing beta(1) integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated beta(1) integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1-mediated R-Ras GAP activity controls cell migration by modulating the activity of beta(1) integrins.     

POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila

Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. Here, we show that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs resulted in roughened or melanized eyes, respectively. These phenotypes were enhanced by co-overexpression of POSH. We found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway.     

Ozone-Induced Cell Death Mediated with Oxidative and Calcium Signaling Pathways in Tobacco Bel-W3 and Bel-B Cell Suspension Cultures

Ozone (O₃)-induced cell death in two suspension-cultured cell lines of tobacco (Nicotiana tabacum L.) derived from Bel-W3 (hyper-sensitive to O₃) and Bel-B (highly tolerant to O₃) varieties were studied. By exposing the newly prepared cell lines to the pulse of ozonized air, we could reproduce the conditions demonstrating the difference in O₃ sensitivity as observed in their original plants, depending on the exposure time. Since O₃-induced acute cell death was observed in the dark, the requirement for photochemical reactions could be eliminated. Addition of several ROS scavengers and chelators inhibited the cell death induced by O₃, indicating that singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radical and redox-active metals such as Fe²⁺ play central roles in O₃-induced acute damages to the cells. As expected, we observed the generation of ¹O₂ and H₂O₂ in the O₃-treated cells using chemiluminescent probes. On the other hand, an NADPH oxidase inhibitor, superoxide dismutase (SOD), and some SOD mimics showed no inhibitory effect. Thiols added as antioxidants unexpectedly behaved as prooxidants drastically enhancing the O₃-induced cell death. It is noteworthy that some ROS scavengers effectively rescued the cells from dying even treated after the pulse of O₃ exposure, confirming the post-ozone progress of ROS-dependent cell death mechanism. Since one of the key differences between Bel-B and Bel-W3 was suggested to be the capacity for ROS detoxification by catalase, the endogenous catalase activities were compared in vivo in two cell lines. As expected, catalase activity in Bel-B cells was ca. 7-fold greater than that in Bel-W3 cells. Interestingly, Ca²⁺ chelators added prior to (not after) the pulse of O₃ effectively inhibited the induction of cell death. In addition, increases in cytosolic Ca²⁺ concentration sensitive to Ca²⁺ chelators, ion channel blockers, and ROS scavengers were observed in the transgenic Bel-W3 cells expressing aequorin, suggesting the action of Ca²⁺ as a secondary messenger initiating the oxidative cell death. The O₃-induced calcium response in Bel-W3 cells was much greater than Bel-B cells. Based on the results, possible pathways for O₃-dependent generation of the lethal level of ROS and corresponding signaling mechanism for induction of cell death were discussed.Key Words: calcium, cell death, Nicotiana tabacum L., ozone, reactive oxygen species     

Preliminary human study for possible alteration of serum immunoglobulin E production in perennial allergic rhinitis with fermented milk prepared with Lactobacillus gasseri TMC0356

The fermented milk prepared with Lactobacillus gasseri TMC0356 was administered at 200 ml per day for 4 weeks to 15 subjects with high serum IgE levels and perennial allergic rhinitis. The serum total IgE concentration was significantly reduced after 28 days' exposure to the fermented milk (P <0.05) compared to that before the intervention. The serum IgE specific to Acari and those to Japanese cedar pollen also significantly declined (P <0.05). T helper 1 (Th1) cells in the composition of their peripheral blood mononuclear cells (PBMCs) significantly increased after 14 days (P <0.01) and after 28 days (P <0.05). These results suggest that the fermented milk prepared with L. gasseri TMC0356 may alter serum IgE concentration, at least partly by enhancement of Th1 immune responses of the subjects with high concentration of serum IgE. However, further studies are still necessary to know the underlying mechanisms by which the tested fermented milk could influence the host immunity.