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41.
42.
Thierry Pillot Anne Barbier Athanase Visvikis Karine Lozac'h Maryvonne Rosseneu Joel Vandekerckhove Gérard Siest 《Protein expression and purification》1996,7(4):407-414
We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH2-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins. 相似文献
43.
Gruber Véronique Berna Patrick P. Arnaud Thierry Bournat Philippe Clément Christèle Mison Dominique Olagnier Béatrice Philippe Laurence Theisen Manfred Baudino Sylvie Bénicourt Claude Cudrey Claire Bloës Carole Duchateau Nathalie Dufour Sylvie Gueguen Catherine Jacquet Séverine Ollivo Catherine Poncetta Christine Zorn Nathalie Ludevid Dolores Van Dorsselaer Alain Verger Robert Doherty Annette Mérot Bertrand Danzin Charles 《Molecular breeding : new strategies in plant improvement》2001,7(4):329-340
A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants. 相似文献
44.
Single-Cell Multiomics: Multiple Measurements from Single Cells 总被引:1,自引:0,他引:1
45.
酸性矿山废水污染的水稻田土壤中重金属的微生物学效应 总被引:20,自引:1,他引:20
采样调查了广东大宝山地区受酸性采矿废水长期污染的亚热带水稻田的土壤理化性质 ,重金属 Cu、Pb、Zn、Cd的全量及其 DTPA浸提量 ,以及微生物生物量及其呼吸活性等指标。利用主成分和逐步回归分析了影响土壤重金属的有效性及其微生物学效应的因素。结果表明 :土壤高含硫 ,强酸性 ,有机碳、全氮较低 ,4种金属的全量普遍超标。DTPA可提取态金属含量较高 ,不仅与其全量呈显著正相关 ,而且与土壤酸度和粘粒含量正相关 ,和 Mn含量负相关。过量的金属显著降低了土壤微生物生物量 C、N、微生物商、生物量 N/全 N比 ,并抑制了微生物呼吸强度和对有机碳的矿化率 ,导致了土壤 C/N比的升高。同时 ,金属对微生物群落及生理代谢指标 ,如微生物生物量 C/N比和代谢商的影响不显著。 DTPA可提取态金属 ,特别是 DTPA- Cu是导致微生物生物量和活性指标变化的主要因素。以有机碳 (或全氮 )为基数的复合微生物指标降低了土壤性质差异造成的干扰 ,较单一指标更能准确指示微生物对金属胁迫的反应。土壤硫没有对金属有效性和微生物指标产生明显影响 ,但其氧化过程可能引起酸化和金属离子的释放 相似文献
46.
Galès G Libert MF Sellier R Cournac L Chapon V Heulin T 《FEMS microbiology letters》2004,240(2):155-162
Although being deionized, filtered and therefore normally deeply oligotrophic, the water from a basin containing irradiating waste presented relatively high bacterial concentrations (ca 10(5) cfu ml(-1)) and biofilm development at its surface and on the walls. This water was characterized by a high concentration of molecular H2 due to water radiolysis, while its electrochemical potential was around +400 mV due the presence of dissolved O2 and active oxygen compounds. This combination of H2 availability and of an oxidant environment is completely original and not described in nature. From surface and wall biofilms, we enumerated the autotrophic populations ( approximately 10(5) bacteria ml(-1)) able to grow in presence of H2 as energy source and CO2 as carbon source, and we isolated the most abundant ones among cultivable bacteria. They efficiently grew on a mineral medium, in the presence of H2, O2 and CO2, the presence of the three gases being indispensable. Two strains were selected and identified using their rrs gene sequence as Ralstonia sp. GGLH002 and Burkholderia sp. GGLH005. In pure culture and using isotope exchange between hydrogen and deuterium, we demonstrated that these strains are able to oxidize hydrogen as energy source, using oxygen as an electron acceptor, and to use carbon dioxide as carbon source. These chemoautotroph hydrogen-oxidizing bacteria probably represent the pioneer bacterial populations in this basin and could be primary producers in the bacterial community. 相似文献
47.
Thierry Brun Jean Péduzzi Manuela Marin Caniça Gérard Paul Pierre Névot Michel Barthélémy Roger Labia 《FEMS microbiology letters》1994,120(1-2):111-117
Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism. 相似文献
48.
49.
The appropriateness of the Amplified Fragment Length Polymorphism (AFLP) technique for investigating Chondrus crispus Stackhouse populations in the Maritime Provinces of Canada was assessed. The AFLP procedure was first subjected to reproducibility
testing and three shortcomings were noted: 1) failure to reproduce band intensity between replicate runs for the same individual
and primer pair; 2) failure of some bands to replicate; 3) lack of reproducibility for complete replicate runs for some individuals
and primer pairs. In the last-mentioned case, the lack of reproducibility resulted in characteristic electropherograms indicative
of weak reactions. These weak runs can be attributed to poor restriction digest/ligation reactions and/or substandard PCR,
these failures ultimately resulting from low and inconsistent DNA quality. We recommend that reproducibility testing should
be completed routinely in studies using the AFLP technique. In the current work, only fragments and individuals that gave
reproducible results were used in subsequent analyses. The AFLP method resulted in highly variable markers within and between
the populations of C. crispus included in this investigation, which prevented successful resolution of population structure. This situation could result
from a lack of suitability for AFLP markers in population genetic studies, and/or too extensive genetic variation for C. crispus populations to be discerned by the AFLP technique. These two possible explanations are discussed.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
50.
Simone Benedetto Véronique Drai-Zerbib Marco Pedrotti Geoffrey Tissier Thierry Baccino 《PloS one》2013,8(12)
The mass digitization of books is changing the way information is created, disseminated and displayed. Electronic book readers (e-readers) generally refer to two main display technologies: the electronic ink (E-ink) and the liquid crystal display (LCD). Both technologies have advantages and disadvantages, but the question whether one or the other triggers less visual fatigue is still open. The aim of the present research was to study the effects of the display technology on visual fatigue. To this end, participants performed a longitudinal study in which two last generation e-readers (LCD, E-ink) and paper book were tested in three different prolonged reading sessions separated by - on average - ten days. Results from both objective (Blinks per second) and subjective (Visual Fatigue Scale) measures suggested that reading on the LCD (Kindle Fire HD) triggers higher visual fatigue with respect to both the E-ink (Kindle Paperwhite) and the paper book. The absence of differences between E-ink and paper suggests that, concerning visual fatigue, the E-ink is indeed very similar to the paper. 相似文献