Abundance and Diversity of Methanotrophs and Propanotrophs in Soils above Yangxin Oil Reservoir,China

Methanotrophs have long been used as an important biological indicator for prospecting of oil and gas, while the indication of propanotrophs in hydrocarbon micro-seep systems is still poorly investigated. In this study, the abundance and diversity of the methanotrophic pmoA gene and the propanotrophic prmA gene as target genes were investigated in soils above Yangxin oil reservoir and Beiguan non-petroliferous area using molecular biological techniques. A total of 14 soil samples were collected at different depths (5, 20, 50, 100, 150, 200 and 250 cm) of two 2.5-m soil profiles located separately within the oil field and the non-petroliferous area for analysis of fluorescent quantitative real-time polymerase chain reaction (RT-PCR) (14 samples) and clone libraries (4 samples). The results demonstrated high presence of the propanotrophic prmA gene ranging from 7.68 × 10⁵ to 2.29 × 10⁷ copies/g dw (gene copies per gram soil of dry weight) in soil from the oil field relative to the non-petroliferous area for which the same measurements yielded results all below detection limit except for the 5-cm sample. On the other hand, oil field soil yielded much lower content of the methanotrophic pmoA gene (below detection limit to 5.6 × 10² copies/g dw) than the non-petroliferous area (1.14 × 10³ copies/g dw to 1.26 × 10⁵ copies/g dw) below 20-cm depth due to influence of biogenic methane, implying that propanotrophs may be better indicator bacteria for prospecting of oil and gas. Almost all pmoA clones of two 50-cm soil samples phylogenetically belonged to Gamma-Proteobacteria and the predominant pmoA OTUs were all uncultured bacteria. All prmA clones of two 5-cm soil samples were derived predominantly from Actinobacteridae (25.7%) and Alpha-Proteobacteria (74.3%), and all dominant prmA OTUs were also clustered with uncultured bacteria. Our results confirm that propanotrophs may be better indicator bacteria for prospecting of oil and gas and enrich the knowledge on diversity of methanotrophs and propanotrophs in the oil field and the non-petroliferous area.     

Expression and production of soluble Mycobacterium tuberculosis H37Rv mycosin-3

Mycobacteria encode five type VII secretion system (T7SS) or ESX for nutrient acquisition and virulence. Mycosins are membrane-anchored components of ESX with serine protease activity but an unidentified substrate range. Establishing the substrate specificity of individual mycosins will help to elucidate individual ESX functions. Mycosin-1 and -3 orthologues from two environmental mycobacterial species, Mycobacterium smegmatis and Mycobacterium thermoresistibile, have been heterologously produced, but mycosins from Mycobacterium tuberculosis (Mtb) remain to be studied. Here we describe the successful production of Mtb mycosin-3 as a first step in investigating its structure and function.     

腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

【目的】通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶Tt Alr底物通道内氨基酸位点A172和S173的功能。【方法】利用定点突变PCR技术构建突变体,通过亲和层析法纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。【结果】通过定点突变PCR成功得到8个突变体,酶学特性分析发现,A172位点突变为丝氨酸(S)后酶蛋白的相对活性有所提升,但含有该位点突变的酶蛋白稳定性均大幅下降;S173位点突变为天门冬氨酸(D)后导致突变体蛋白的最适反应温度提升了15°C,半衰期大幅延长,但相对活性明显下降。【结论】丙氨酸消旋酶Tt Alr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。     

Study of the molecular mechanism of interleukin-2 mutein D10 binding to IL-2 receptors by molecular simulations

Interleukin-2 (IL-2) was originally used therapeutically as an immune stimulatory agent due to its characteristics of immune regulation. Previous work showed that binding of IL2 to its α-subunit receptor induced low blood pressure, vascular leak syndrome and cardiac toxicity. It was reported that there would be no side effects if the mutein of IL-2 could bind directly with the IL-2β/IL-2Rγ complex, and without mediating with IL-2Rα. With the aim to understand the differences in the binding affinity between IL-2 and the mutein to IL-2Rβ/IL-2Rγ complex at an atomic level, we constructed the theoretical binding models of a wild type and a mutated type (D10) of IL-2 utilising homology modelling. We analysed the interactive differences between the two systems after the MD simulations were completed. Our results suggested that (1) the mutation Q74H caused the conformational change of the loop and led to a new binding mode, which consequently caused an increased binding affinity for IL-2Rβ; (2) the residue mutation of I92F provided more favourable interactions; (3) the mutation of R81D provided an increased binding affinity, a contribution by both direct and indirect interactions. The newly designed mutants, D10_D81E, D10_F92 W and D10_N119E, were predicted to have a better potency, especially at the residue 119, which was firstly found to improve the binding activity of IL-2Rγ. These results are expected to facilitate the discovery and rational design of novel IL-2 stimulatory agents, a potential treatment for cancer patients.     

Design,synthesis and biological evaluation of novel tetrahydroisoquinoline derivatives as P-glycoprotein-mediated multidrug resistance inhibitors

Multidrug resistance (MDR) is one of the main obstacles of clinical chemotherapy. A great deal of research shows that the occurrence of drug resistance in various malignant tumors is closely related to the expression of P-glycoprotein (P-gp) on the surface of the cell membrane. In this paper, based on the structure-activity relationship of phenylethyl tetrahydroisoquinoline, we choose tariquidar as the lead compound for the design and synthesis of 17 novel tetrahydroisoquinoline P-gp inhibitors. Additionally, in vitro and in vivo cytotoxicity assays and reversed MDR activity assays were evaluated. Among them, compound 3 had a good reversal of MDR activity and the reversal mechanism study of it was carried out. All of these results demonstrated that compound 3 was considered to be a promising P-gp-mediated MDR reversal candidate.     

种衣剂对低温下棉花幼苗光合特性的影响

为研究种衣剂对低温下棉花幼苗光合特性的影响,以"新陆早57号"为材料,采用室内盆栽培养法,温度设置为10℃和18℃,胁迫时间为0、1、2、3d,分别从光合色素、光合气体交换参数以及叶绿素荧光动力学三方面探讨种衣剂对棉花幼苗低温伤害的缓解作用。结果表明:(1)与未包衣棉苗相比,种衣剂可提高低温胁迫下棉花幼苗的叶绿素a、b及类胡萝卜素含量,但在常温下种衣剂对棉苗的光合色素含量均无显著影响。(2)与未包衣棉苗相比,包衣棉苗在10℃和18℃处理3d后,净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(G_s)分别依次提高33.71%、52.00%、96.63%和38.57%、55.17%、103.42%,胞间二氧化碳浓度(C_i)分别降低了15.15%、11.12%。(3)与未包衣棉苗相比,包衣棉苗在10℃和18℃处理3d后,单位反应中心吸收的光能(ABS/RC)分别增加了0.41%、1.56%,捕获用于还原Q_A的能量(TR_o/RC)和捕获用于电子传递的能量(ET_o/RC)分别增加了2.97%、0.47%和4.27%、1.67%,反应中心用于电子传递的激子与用来推动Q_A还原激子的比率(ψ_o)分别增加了0.18%、2.16%,吸收的光能用于电子传递的量子产额(Φ_(Eo))和性能指数(PI_(ABS))分别增加了2.25%、2.42%和19.32%、5.94%。研究认为,种衣剂包衣处理可以提高低温下棉花幼苗的光合色素含量、调节气孔开度,增强CO_2的固定能力和利用率,提高光合吸收及捕获的能量,并诱导光合能量合理分配,维持光合器官对光能的吸收、捕获、转化状况,从而减轻低温胁迫带来的伤害。     

Human Umbilical Cord Mesenchymal Stem Cells Protect Against SCA3 by Modulating the Level of 70 kD Heat Shock Protein

Spinocerebellar ataxia 3 (SCA3), which is a progressive neurodegenerative disease, is currently incurable. Emerging studies have reported that human umbilical cord mesenchymal stem cells (HUC-MSCs) transplantation could be a promising therapeutic strategy for cerebellar ataxias. However, few studies have evaluated the effects of HUC-MSCs on SCA3 transgenic mouse. Thus, we investigated the effects of HUC-MSCs on SCA3 mice and the underlying mechanisms in this study. SCA3 transgenic mice received systematic administration of 2 × 10⁶ HUC-MSCs once per week for 12 continuous weeks. Motor coordination was measured blindly by open field tests and footprint tests. Immunohistochemistry and Nissl staining were applied to detect neuropathological alternations. Neurotrophic factors in the cerebellum were assessed by ELISA. We used western blotting to detect the alternations of heat shock protein 70 (HSP70), IGF-1, mutant ataxin-3, and apoptosis-associated proteins. Tunel staining was also used to detect apoptosis of affected cells. The distribution and differentiation of HUC-MSCs were determined by immunofluorescence. Our results exhibited that HUC-MSCs transplantation significantly alleviated motor impairments, corresponding to a reduction of cerebellar atrophy, preservation of neurons, decreased expression of mutant ataxin-3, and increased expression of HSP70. Implanted HUC-MSCs were mainly distributed in the cerebellum and pons with no obvious differentiation, and the expressions of IGF-1, VEGF, and NGF in the cerebellum were significantly elevated. Furthermore, with the use of HSP70 analogy quercetin injection, it demonstrated that HSP70 is involved in mutant ataxin-3 reduction. These results showed that HUC-MSCs implantation is a potential treatment for SCA3, likely through upregulating the IGF-1/HSP70 pathway and subsequently inhibiting mutant ataxin-3 toxicity.     

Pollen morphology of Quercus sect. Ilex and its relevance for fossil pollen identification in southwest China

Pollen grains of 16 modern species of Quercus were examined by scanning electron microscopy (SEM) and light microscopy (LM). The focus of this investigation was on a small group (c. eight species) of alpine, sclerophyllous oaks that may form a monophyletic group within sect. Ilex. The data were combined with published data for Polar axis (P) and Equatorial diameter (E) for more than 80 species. Based on pollen morphology Quercus pollen grains were divided into three types: (1) The Cyclobalanopsis type is smallest (generally < 23 μm) in size, with P/E ratios of approximately 1.08 and a psilate or fine-grained surface; (2) Quercus sect. Ilex type is 23–29 μm, the P/E ratio is approximately 1.24 (prolate) and it has a rugulate pollen surface; (3) the Quercus sect. Quercus type is generally > 29 μm in size, has a P/E ratio of approximately 1.04, and a clearly verrucate pollen surface. Based on a comparison with modern pollen size and ornamentation, fossil pollen grains of the Quaternary Qinghai Lake core can be assigned to the Chinese southwest mountain oak group of Quercus sect. Ilex (in particular Quercus semecarpifolia). In addition, we show that the pollen percentages of Quercus rapidly increased to the highest level during the glacial period. Using SEM for the identification of fossil Quercus pollen grains is essential to ensure the accuracy of differentiating evergreen Quercus types (sect. Ilex versus sect. Cyclobalanopsis) during the last glacial period in southwest China.