Preparation and characterization of magnetic microspheres for the purification of interferon alpha-2b

Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN alpha-2b monoclonal antibodies (mAb) were selected for the purification of interferon alpha-2b (IFN alpha-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN alpha-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN alpha-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 x 10(8) IU/mg. Identity and molecular mass of purified IFN alpha-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.     

Effects of Forest Regrowth and Urbanization on Ecosystem Carbon Storage in a Rural–Urban Gradient in the Southeastern United States

Forest regrowth after cropland abandonment and urban sprawl are two counteracting processes that have influenced carbon (C) sequestration in the southeastern United States in recent decades. In this study, we examined patterns of land-use/land-cover change and their effect on ecosystem C storage in three west Georgia counties (Muscogee, Harris, and Meriwether) that form a rural–urban gradient. Using time series Landsat imagery data including MSS for 1974, TM for 1983 and 1991, and ETM for 2002, we estimate that from 1974 to 2002, urban land use in the area has increased more than 380% (that is, 184 km²). Most newly urbanized land (63%) has been converted from forestland. Conversely, cropland and pasture area has decreased by over 59% (that is, 380 km²). Most of the cropland area was converted to forest. As a result, the net change in forest area was small over the past 29 years. Based on Landsat imagery and agricultural census records, we reconstructed an annual gridded data set of land-cover change for the three counties for the period 1850 to 2002. These data sets were then used as input to the Terrestrial Ecosystem Model (TEM) to simulate land-use effects on C fluxes and storage for the study area. Simulated results suggest that C uptake by forest regrowth (approximately 23.0 g C m⁻² y⁻¹) was slightly greater than the amount of C released due to deforestation (approximately 18.4 g C m⁻² y⁻¹), thus making the three counties a weak C sink. However, the relative importance of different deforestation processes in this area changed significantly through time. Although agricultural deforestation was generally the most important C-release process, the amount of C release attributable to urbanization has increased over time. Since 1990, urbanization has accounted for 29% of total C loss from the study area. We conclude that balancing urban development and forest protection is critically important for C management and policy making in the southeastern United States.     

经血管灌流生长抑素对大鼠离体胃运动的抑制作用

采用血管灌流大鼠离体胃模型,探讨生长抑素对胃运动的影响。结果表明：（１）生长抑素能明显抑制胃窦自发和胃动素兴奋的胃运动;（２）生长抑素可抑制离体胃内源性胃泌素释放;（３）抗生长抑素血清和前列腺素合成酶抑制剂消炎痛可阻断生长抑素对胃窦运动的抑制作用。上述结果提示：生长抑素的抑制作用除通过直接作用于生长抑素受体外,还可能通过胃窦局部前列腺素介导来抑制胃的运动。     

Enhanced chemiluminescence enzyme‐linked immunoassay for the determination of DNA methyltransferase 1 in human serum

The occurrence of many diseases is closely related to the high expression of DNA methyltransferase 1 (DNMT1). However, most studies are focused on the detection of DNMT1 activity, a few are concerned with the detection of DNMT1 content. In this study, we developed a simple and highly sensitive chemiluminescence (CL) assay for the detection of DNMT1 content. In this method, anti‐DNMT1 monoclonal antibody was coated on a polystyrene microplate to capture DNMT1. Then anti‐DNMT1 polyclonal antibody and goat anti‐rabbit immunoglobulin G with horseradish peroxidase (IgG‐HRP) were respectively added to combine with captured DNMT1 to form a sandwich structure. Finally, the HRP could catalyze CL substrate and achieve CL signal response. Based on this novel sensitive strategy, the recovery percents were in the ranges from 71.5% to 91.0%. The precision of intra‐assays and inter‐assays were 5.45%–11.29% and 7.03%–11.25%, respectively. The method was successfully applied for the determination of DNMT1 in human serum. The detection results of serum samples showed that the proposed assay had a high correlation with enzyme‐linked immunosorbent assay (ELISA) kit. Compared with the ELISA kit (limit of detection = 0.1 ng/mL), the method has a lower limit of detection of 0.042 ng/mL. Therefore, our method has the potential for the detection of DNMT1 content in clinical diagnosis.     

簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。     

丝状真菌瑞氏木霉外源基因表达系统的构建

采用PCR技术体外扩增获得了瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅰ (CBHⅠ )启动子和终止子序列 .并以大肠杆菌质粒pUC1 9为骨架 ,在该启动子和终止子序列间加入多克隆位点 ,构建了瑞氏木霉强表达整合型载体pTRIL .以质粒pAN7 1为模板 ,体外扩增了带有潮霉素磷酸转移酶(hph)基因的DNA片段 ,将hph插入pTRIL的cbh1启动子和终止子序列之间 ,构建了Pcbh1 hph Tcbh1表达盒 .用此表达盒转化瑞氏木霉C30原生质体 ,在潮霉素平板上得到 1 5株抗性转化子 .对其中的H1转化子进行了PCR和Southern印迹分析 ,证实hph基因确实整合到转化子染色体DNA上 ,并在Pcbh1 启动子控制下进行高效表达 .转化子H1对潮霉素抗性达 1 5 0mg L ,比出发菌株提高 2倍 .瑞氏木霉强表达整合型载体pTRIL的构建成功为开展瑞氏木霉分子生物学研究以及进一步的工程菌株构建工作奠定了基础