Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764

We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ ¹H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 10³ M^?1 s^?1) is less than stellar.     

Carbon sequestration in soil in a semi‐natural Miscanthus sinensis grassland and Cryptomeria japonica forest plantation in Aso,Kumamoto, Japan

Although Miscanthus sinensis grasslands (Misc‐GL) and Cryptomeria japonica forest plantations (Cryp‐FP) are proposed bioenergy feedstock systems, their relative capacity to sequester C may be an important factor in determining their potential for sustainable bioenergy production. Therefore, our objective was to quantify changes in soil C sequestration 47 years after a Misc‐GL was converted to a Cryp‐FP. The study was conducted on adjacent Misc‐GL and Cryp‐FP located on Mt. Aso, Kumamoto, Japan. After Cryp‐FP establishment, only the Misc‐GL continued to be managed by annual burning every March. Mass C and N, δ¹³C, and δ¹⁵N at 0–30 cm depth were measured in 5 cm increments. Carbon and N concentrations, C:N ratio, δ¹³C, and δ¹⁵N were measured in litter and/or ash, and rhizomes or roots. Although C input in Misc‐GL by M. sinensis was approximately 36% of that in Cryp‐FP by C. japonica, mean annual soil C sequestration in Misc‐GL (503 kg C ha^?1 yr^?1) was higher than that in Cryp‐FP (284 kg C ha^?1 yr^?1). This was likely the result of larger C input from aboveground litter to soil, C‐quality (C:N ratio and lignin concentration in aboveground litter) and possibly more recalcitrant C (charcoal) inputs by annual burning. The difference in soil δ¹⁵N between sites indicated that organic C with N had greater cycling between heterotrophic microbes and soil and produces more recalcitrant humus in Misc‐GL than in Cryp‐FP. Our data indicate that in terms of soil C sequestration, maintenance of Misc‐GL may be more advantageous than conversion to Cryp‐FP in Aso, Japan.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.     

中国齿裂菌属研究III.

报道齿裂菌属的2个新种：生于青冈Cyclobalanopsis glauca (Thunb.) Oerst.及绵石栎 Lithocarpushenryi (Seem.)Rehd. etWils.上的黄山齿裂菌Coccomyces huangshanensis Y.R. Lin & Z. Z. Li sp. nov.和生于青冈上的大齿裂菌 C. magnus Y. R. Lin & Z. Z. Li sp. nov.;2个中国新记录种：小齿裂菌 C. leptideus (Fr. : Fr.) Erikss. 和显缘齿裂菌 C. limitatus (Berk. & Curt.) Sacc.。对新种作了拉丁文及汉文描述和图解。模式标本存于安徽农业大学森林保护教研室 (AAUFP）。     

激光联合氨甲环酸、谷胱甘肽及维生素C 治疗黄褐斑的临床观察

目的：探讨黄褐斑应用激光联合氨甲环酸、谷胱甘肽及维生素C 治疗的临床效果。方法：选择2012 年6 月~2014 年6 月期 间我院皮肤科门诊收治的黄褐斑女性患者117 例,根据随机数字表将其平均分为三组,即激光组、药物组、联合治疗组各39 例。 激光组单纯应用激光治疗,药物组应用氨甲环酸、谷胱甘肽及维生素C治疗,联合治疗组应用激光联合氨甲环酸、谷胱甘肽及维 生素C 治疗。对比三组治疗前后黄褐斑面积及黄褐斑颜色评分、临床疗效及治疗后不良反应。结果：联合组治疗后黄褐斑面积及 黄褐斑颜色评分显著低于激光组、药物组及治疗前（P<0.05）。联合组治疗的总有效率显著高于激光组及药物组（P<0.05）。药物组 不良反应发生率为2.56%,其他两组未见明显不良反应。结论：激光联合氨甲环酸、谷胱甘肽及维生素C 治疗黄褐斑的疗效确切, 可以有效改善黄褐斑面积及颜色,安全性佳,适于临床应用。     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

内蒙古大针茅草原常见植物在不同土地利用方式下的固碳相关属性

为了探究植物与固碳相关属性在不同功能群、器官和物种间的差异, 以及这些属性对不同土地利用方式的响应, 2012年8月对内蒙古大针茅草原的4个样地(长期无干扰样地、长期自由放牧样地、4年围封样地和4年围封割草样地)进行了群落调查, 并采集样地中的常见植物, 测定了与植物固碳相关的属性, 包括全碳含量、全氮含量、碳氮比、纤维素含量、木质素含量和酸性洗涤纤维含量等。以常见植物为对象, 在功能群水平研究了各土地利用方式下全碳含量、全氮含量和碳氮比的差异; 针对各样地的共有种——糙隐子草(Cleistogenes squarrosa)、大针茅(Stipa grandis)、冷蒿(Artemisia frigida)、羊草(Leymus chinensis)和猪毛菜(Salsola collina), 从物种和器官水平分析了不同土地利用方式下植物的固碳相关属性。结果表明: 大针茅草原植物不同功能群、物种和器官间的固碳相关属性存在极显著差异, 不同土地利用方式下大针茅草原不同功能型、物种和器官的固碳相关属性有显著差异。与其他利用方式相比, 4年围封割草对植物与固碳相关属性的影响最为明显, 功能群、器官和物种水平的植物氮含量均有下降。糙隐子草和猪毛菜的这些属性对长期自由放牧的响应敏感, 且方向相反。