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251.
A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing
phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering.Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The
expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant.
The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the
same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression
of other small peptides. 相似文献
252.
S. H. Gan R. Ismail 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,759(2):287
An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36±12.53% and 93.52±7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study. 相似文献
253.
Impact of fumigants on soil microbial communities. 总被引:12,自引:0,他引:12
A M Ibekwe S K Papiernik J Gan S R Yates C H Yang D E Crowley 《Applied and environmental microbiology》2001,67(7):3245-3257
Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact. 相似文献
254.
Rapid Identification of Probiotic Lactobacillus Biosurfactant Proteins by ProteinChip Tandem Mass Spectrometry Tryptic Peptide Sequencing 总被引:1,自引:0,他引:1 下载免费PDF全文
Gregor Reid Bing Siang Gan Yi-Min She Werner Ens Scot Weinberger Jeffrey C. Howard 《Applied microbiology》2002,68(2):977-980
A novel ProteinChip-interfaced tandem mass spectrometer was employed to identify collagen binding proteins from biosurfactant produced by Lactobacillus fermentum RC-14. On-chip tryptic digestion of the captured collagen binding proteins resulted in rapid sequence identification of five novel tryptic peptide sequences via collision-induced dissociation tandem mass spectrometry. 相似文献
255.
256.
Gene expression in the developing mouse retina by EST sequencing and microarray analysis 总被引:9,自引:0,他引:9 下载免费PDF全文
257.
人睫状神经营养因子的原核表达,纯化及其生物效应 总被引:2,自引:0,他引:2
人睫状神经营养因子(hCNTF)克隆入pBV220中,在DH5α菌株中表达,重组蛋白以包含体的形式存在,表达量为菌体总蛋白的50%左右。经比较发现用2mol/L脲洗涤包含体可溶解大量可溶性细菌蛋白,且包含体损失较小。在高浓度变性剂条件下进行sepharcylS-200凝胶过滤,解决了纯化中hCNTF易聚合的问题,在低浓度变性剂条件下进行DEAE离子交换,有利于蛋白活性的保持。经两步纯化后得到均一性hCNTF,纯度达95%以上。在自然状态下使hCNTF复性。纯化复性后的hCNTF对无血清培养的鸡胚背根节神经元和脊髓腹角运动神经元有明显的维持存活和促进生长发育的生物效应。 相似文献
258.
259.
Abstract: Elevated concentrations of extracellular K+ increased inositol phosphate accumulation in primary cultures of chick retinal photoreceptors and multipolar neurons. K+ -evoked stimulation of inositol phosphate accumulation was greater in photoreceptor-enriched cell cultures than in cultures where multipolar neurons were the predominant cell type. Destroying multipolar neurons, but not photoreceptors, with kainic acid and N -methyl- d -aspartate did not reduce the K+ -evoked stimulation of inositol phosphate accumulation. Both of these observations indicate that the observed effects occur in photoreceptor cells. The K+ -evoked stimulation of inositol phosphate accumulation was blocked by omitting Ca2+ from the incubation medium or by adding the dihydropyridine-sensitive Ca2+ -channel antagonists, nitrendipine and nifedipine. Bay K 8644, a dihydropyridine agonist, stimulated inositol phosphate accumulation and enhanced the effect of K+ . ω-Conotoxin GVIA, an inhibitor of N-type Ca2+ channels, had no significant effect on K+ -stimulated inositol phosphate accumulation. Pretreatment with pertussis toxin neither blocked K+ -evoked inositol phosphate accumulation nor altered the inhibitory effect of nifedipine. K+ -evoked inositol phosphate accumulation appears to reflect activation of phosphatidylinositol-specific phospholipase C, as it is inhibited by U-73122. These results indicate that Ca2+ influx through voltage-gated, dihydropyridine-sensitive channels activates phospholipase C in photoreceptor inner segments and/or synaptic terminals. 相似文献
260.