Rpf蛋白：一种激活休眠状态放线菌的复苏促进因子

放线菌能产生多样性丰富的小分子化合物,但大多数放线菌因为处于“活的尚未培养”状态而无法分离培养。造成“活的尚未培养”状态的原因之一可能是由于一些环境因素,例如有机物、重金属、抗生素等胁迫使细胞处于休眠保护状态,直到遇到适宜的条件才能继续复苏生长。复苏促进因子（Resuscitation-Promoting Factor,Rpf）是由某些放线菌分泌的一类蛋白质,首次在藤黄微球菌（Micrococcus luteus）中发现,之后人们对Rpf蛋白的功能和分布给予了更多的关注。Rpf蛋白能促进一些休眠的革兰氏阳性细菌复苏,这为“活的尚未培养”放线菌的分离培养提供了可能性。同时,针对一些致病放线菌物种,开发Rpf蛋白抑制物为相关疾病的治疗也提供了一条新的途径。基于此,本文对Rpf蛋白的结构组成、特征功能、作用机制及应用前景进行了简要的概述。     

黄土丘陵区人工柠条种植年限和坡位对土壤团聚体稳定性的影响

柠条(Caragana korshinskii Kom.)种植是黄土高原地区生态环境建设中重要的人工植被恢复措施。选择黄土丘陵区条带种植15、25和35年的柠条坡地,以荒草坡地为对照,运用Le Bissonnais法分析柠条种植不同年限和坡位对0—40 cm土层团聚体分布及其稳定性的影响。结果表明:长时间柠条种植对土壤团聚体稳定性的影响主要在0—20 cm土层;不同处理下土壤大团聚体(>0.25 mm)含量总体表现为柠条35年>柠条25年>柠条15年>荒草地,表明柠条种植年限增加促进了大团聚体的形成。从坡面尺度看,柠条平均重量直径整体表现为坡下>坡上>坡中;在上坡的柠条35年样地具有最大值(3.08 mm),但在下坡荒草地显著高于柠条林地(P<0.05)。基于相对消散指数和相对机械破碎指数,上中坡土壤团聚体均对消散作用和机械破碎作用较敏感,而下坡的下层土壤团聚体对破碎作用更敏感。冗余分析表明,土壤有机碳和粘粒含量与柠条平均重量直径呈显著正相关关系(P<0.05)。种植年限是影响土壤团聚体稳定性的主要因素,解释了30.4%的变异;其次是土层...     

Northern richness and southern poverty: contrasting genetic footprints of glacial refugia in the relictual tree Sciadopitys verticillata (Coniferales: Sciadopityaceae)

Sciadopitys verticillata is amongst the most relictual of all plants, being the last living member of an ancient conifer lineage, the Sciadopityaceae, and is distributed in small and disjunct populations in high rainfall regions of Japan. Although mega‐fossils indicate the persistence of the species within Japan through the Pleistocene glacial–interglacial cycles, how the species withstood the colder and drier climates of the glacials is not well known. The present study utilized phylogeography and palaeodistribution modelling to test whether the species survived within pollen‐based coastal temperate forest glacial refugia or within previously unidentified refugia close to its current range. Sixteen chloroplast haplotypes were found that displayed significant geographical structuring. Unexpectedly, northern populations in central Honshu most distant from coastal refugia had the highest chloroplast diversity and were differentiated from the south, a legacy of glacial populations possibly in inland river valleys close to its current northern range. By contrast, populations near putative coastal refugia in southern Japan, harboured the lower chloroplast diversity and were dominated by a single haplotype. Fragment size polymorphism at a highly variable and homoplasious mononucleotide repeat region in the trnT‐trnL intergenic spacer reinforced the contrasting patterns of diversity observed between northern and southern populations. The divergent histories of northern and southern populations revealed in the present study will inform the management of this globally significant conifer. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 263–277.     

Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway

In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.     

Stereoselective synthesis of a novel 2-aza-7-oxabicyclo[3.3.0]octane as neurokinin-1 receptor antagonist

A novel neurokinin-1 receptor antagonist, (±)-(1R^*,3S^*,4S^*,5S^*)-4-[(N-(2-methoxy-5-trifluoromethoxybenzyl)amino]-3-phenyl-2-aza-7-oxabicyclo[3.3.0]octane (1), was synthesized stereoselectively using Padwa’s intramolecular 1,3-dipolar cycloaddition methodology as the key step. Compound (±)-1 showed high affinity for the NK-1 receptors in human IM-9 cells with an IC₅₀ value of 0.22 nM. This new structural scaffold demonstrated significant in vivo antagonistic activity in the guinea pig ureter capsaicin-induced plasma extravasation model with an ED₅₀ value of 1–10 mg/kg, po.     

The promoter of rbcS in a C3 plant (rice) directs organ-specific,light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding -glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.     

Effects of ligand binding on the stability of aldo–keto reductases: Implications for stabilizer or destabilizer chaperones

Ligands such as enzyme inhibitors stabilize the native conformation of a protein upon binding to the native state, but some compounds destabilize the native conformation upon binding to the non‐native state. The former ligands are termed “stabilizer chaperones” and the latter ones “destabilizer chaperones.” Because the stabilization effects are essential for the medical chaperone (MC) hypothesis, here we have formulated a thermodynamic system consisting of a ligand and a protein in its native‐ and non‐native state. Using the differential scanning fluorimetry and the circular dichroism varying the urea concentration and temperature, we found that when the coenzyme NADP⁺ was absent, inhibitors such as isolithocholic acid stabilized the aldo–keto reductase AKR1A1 upon binding, which showed actually the three‐state folding, but destabilized AKR1B10. In contrast, in the presence of NADP⁺, they destabilized AKR1A1 and stabilized AKR1B10. To explain these phenomena, we decomposed the free energy of stabilization (ΔΔG) into its enthalpy (ΔΔH) and entropy (ΔΔS) components. Then we found that in a relatively unstable protein showing the three‐state folding, native conformation was stabilized by the negative ΔΔH in association with the negative ΔΔS, suggesting that the stabilizer chaperon decreases the conformational fluctuation of the target protein or increase its hydration. However, in other cases, ΔΔG was essentially determined by the delicate balance between ΔΔH and ΔΔS. The proposed thermodynamic formalism is applicable to the system including multiple ligands with allosteric interactions. These findings would promote the development of screening strategies for MCs to regulate the target conformations.     

Screening of integrin-binding peptides from the laminin α4 and α5 chain G domain peptide library

Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin α4 and α5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin β1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3β1- and α6β1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.     

Fatty liver induced by free radicals and lipid peroxidation

An excessive accumulation of fat in the liver leads to chronic liver injury such as non-alcoholic fatty liver disease (NAFLD), which is an important medical problem affecting many populations worldwide. Oxidative stress has been implicated in the pathogenesis of NAFLD, but the exact nature of active species and the underlying mechanisms have not been elucidated. It was previously found that the administration of free radical-generating azo compound to mice induced accumulation of fat droplet in the liver. The present study was performed aiming at elucidating the changes of lipid classes and fatty acid composition and also measuring the levels of lipid peroxidation products in the liver induced by azo compound administration to mouse. The effects of azo compound on the liver were compared with those induced by high fat diet, a well-established cause of NAFLD. Azo compounds given to mice either by intraperitoneal administration or by dissolving to drinking water induced triacylglycerol (TG) increase and concomitant phospholipid decrease in the liver, whose pattern was quite similar to that induced by high fat diet. Lipid peroxidation products such as hydroxyoctadecadienoic acid and hydroxyeicosatetraenoic acid were increased in the liver in association with the increase in TG. These results show that free radicals as well as high fat diet induce fatty liver by similar mechanisms, in which lipid peroxidation may be involved.