人热休克蛋白70的原核高效表达

将人热休克蛋白基因hsp70片段克隆到高效原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序。将该重组表达载体转化大肠杆菌DH50α,用IPTG在不同温度及时间下进行诱导表达。收集细菌,菌体裂解后进行SDS-PAGE及Western blot检测,并以凝胶薄层扫描分析表达水平。结果表明,成功地构建了含人hsp70基因的表达载体pMAL-c2X／hsp70,该载体能在大肠杆菌中表达相对分子质量为110000并具有抗原活性的融合蛋白;改变诱导温度和时间,目的蛋白表达总量及可溶性部分所占比例不同。对人hsp70基因的克隆、表达,并对其进行表达条件的优化,为研究HSP70的结构、功能与临床应用提供了必要条件。     

Ammonium,nitrate, and urea play different roles for lipid accumulation in the nervonic acid—producing microalgae <Emphasis Type="Italic">Mychonastes afer</Emphasis> HSO-3-1

Producing valuable coproducts from oleaginous microalgae is an option to reduce the total cost of biofuel production. Here, the influence of nitrogen sources on biomass yield and lipid accumulation of a newly identified oleaginous green microalgal strain, Mychonastes afer HSO-3-1, was evaluated. Carbon assimilation and the following lipid biosynthesis of M. afer were inhibited to some extent under weak acidic conditions (6 < pH < 7) and any of the tested nitrogen source. The highest lipid productivity of 50.7 mg L^?1 day^?1 was achieved with a 17.6 mM nitrogen supplement in the form of urea. The cell polar lipid content was significantly higher than triacylglycerol (TAG), and saturated palmitic acid (C16:0) occupied a dominant position in the fatty acid profiles while culturing M. afer in acidic medium with NH₄ ⁺ as the nitrogen source. Under neutral conditions, the lipid productivities of M. afer cultivated in media containing 17.6 mM of NaNO₃, NH₄Cl, and NH₄NO₃ were 76.2, 77.5, and 79.0 mg L^?1 day^?1, respectively. The greatest TAG content (58.56%) of total lipids was obtained when NaNO₃ was used as the nitrogen source. There was no significant difference in the fatty acid composition of M. afer cells when they were cultivated in neutral media supplemented with NaNO₃, urea, NH₄Cl, and NH₄NO₃. Therefore, NH₄ ⁺ was not a suitable nitrogen source for M. afer cultivation due to the additional labor, working procedures, and alkali required to adjust the medium pH. Considering that using urea as nitrogen source could reduce the cost of nutrient salts substantially and urea can be taken up and utilized by most microalgae, it is a preferred nitrogen source. The major properties of biodiesel derived from M. afer HSO-3-1 met biodiesel quality, and nervonic acid concentrations remained at approximately 3.0% of total fatty acids.     

RNA polymerase pushing

Molecular motors can exhibit Brownian ratchet or power stroke mechanisms. These mechanistic categories are related to transition state position: An early transition state suggests that chemical energy is stored and then released during the step (stroke) while a late transition state suggests that the release of chemical energy rectifies thermally activated motion that has already occurred (ratchet). Cellular RNA polymerases are thought to be ratchets that can push each other forward to reduce pausing during elongation. Here, by constructing a two-dimensional energy landscape from the individual landscapes of active and backtracked enzymes, we identify a new pushing mechanism which is the result of a saddle trajectory that arises in the two-dimensional energy landscape of interacting enzymes. We show that this mechanism is more effective with an early transition state suggesting that interacting RNAPs might translocate via a power stroke.     

湖南不同地区小菜蛾对药剂敏感性比较

采用浸叶法在室内测定了湖南长沙和怀化地区小菜蛾Plutella xylostella(L.)田间种群对10种药剂的敏感性。结果表明:湖南长沙和怀化地区田间小菜蛾除对丁醚脲和BT制剂仍处于敏感(抗性倍数<3)状态外,对其它8种药剂产生了不同程度的抗药性,其中以长沙地区小菜蛾田间种群对高效氯氰菊酯抗性倍数最高(抗性倍数为33.58)。长沙和怀化种群对药剂的相对毒力倍数以巴丹最低(1.20)而以多杀菌素最高(2.59)。     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.     

Molecular cytogenetic characterization and disease resistance observation of wheat-Dasypyrum breviaristatum partial amphiploid and its derivatives

A wheat-Dasypyrum breviaristatum partial amphiploid and its derivatives were analyzed by molecular cytological observation and tested for disease resistance in order to evaluate the potential use of the D. breviaristatum for wheat improvement. A fertility-improved partial amphiploid, TDH-2, was produced from the selfing population of Triticum aestivum cv. Chinese spring (CS)-D. breviaristatum amphiploid. Based on the results obtained from genomic in situ hybridization (GISH) and seed protein electrophoresis, we found the presence of fourteen D. breviaristatum chromosomes and the absence of D genome in TDH-2, indicating that the genomic composition of TDH-2 was AABBV(b)V(b). GISH analysis on BC(1)F(4) progenies of TDH-2xwheat demonstrated that alien D. breviaristatum chromosomes or segments were frequently transmitted. A survey of diseases resistance revealed that powdery mildew resistance from D. breviaristatum was totally expressed, however, the expression of stripe rust resistance from D. breviaristatum was dependent on the wheat background. The comparison of polymerase chain reaction (PCR), which was carried out using molecular marker SCAR(1400) linked to Pm21 D. villosum-derived powdery mildew resistance gene, suggested that D. breviaristatum possessed new resistance gene(s) different from that in D. villosum. The present study showed that the partial amphiploid TDH-2 and its derivatives could serve as novel sources for transfer of disease resistance genes to wheat.     

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

Pre-treating donor cells before somatic cell nuclear transfer (SCNT, 'cloning') may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.     

长裙竹荪抑菌活性物质的分离纯化及其抑菌效果

长裙竹荪Dictyophora indusiata是珍贵的食药用真菌,具有很强的抑菌作用,在天然防腐剂开发方面具有广阔的应用前景。本研究以长裙竹荪的抑菌活性为指标,通过萃取、3次不同流动相的硅胶柱层析、1次反相柱层析和薄层层析法对竹荪提取物进行分离纯化,得到一个抗菌活性强的单体化合物。根据核磁共振波谱等数据分析,推断该化合物为间苯三酚。以巨大芽孢杆菌和肠炎沙门氏菌为供试菌,用平板打孔法及原位抑菌法测定该化合物的抑菌效果,结果表明：该化合物对这两种菌有很强的抑制作用,半抑制浓度分别为83.06μg/mL和51.58μg/mL。本研究首次从长裙竹荪中获得具有抗菌活性的单体化合物间苯三酚,为竹荪天然抗菌物质的开发提供理论依据。     

Tolerance to, and uptake and degradation of 2,4,6-trinitrotoluene (TNT) are enhanced by the expression of a bacterial nitroreductase gene in Arabidopsis thaliana

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C.1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The obtained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the non-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mM TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when theywere floated on TNT solution. HPLC analysis showed that the peak due to TNT taken upinto plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indicates that the expression of bacterial NTR improved the capability of plants to degrade TNT.