Construction of recombinant plasmids containing rat thyroglobulin mRNA sequences

Two plasmids containing rat thyroglobulin cDNA sequences have been constructed and characterized. A plasmid with a 500-bp insert (pRT6) was isolated and identified as thyroglobulin-specific on the basis of the tissue specificity of the inserted sequence and of its ability to retain thyroglobulin mRNA on a nitrocellulose filter. The cDNA insert in pRT6 was subsequently used to screen a rat thyroid cDNA library constructed with large cDNA. A plasmid was found containing a 1700-bp insert. The polarity and the fidelity of the insert is demonstrated by S1 mapping.     

Recent progresses in gene delivery-based bone tissue engineering

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.     

应用于DNA分子转移和分子杂交实验的二偶氮苄氧甲基(DBM)-滤纸性质的研究

DBM-滤纸与硝基纤维素一样,近年来广泛地用作DNA或RNA分子的载体来进行分子杂交实验。但是在进行小分子DNA的分子和杂交实验中,以及需要用几种不同的分子探针在同一个DNA载体上反复地进行分子杂交时,DBM-滤纸显示了更大的优越性。用小球菌核酸酶水解鸡红血球细胞核,抽提出DNA并用[~(32)P-γ]ATP和T_4多核苷酸激酶制备成5’末端标记的DNA分子。经2%琼脂糖电泳,DNA分子按染色质的核小体结构的一系列不同长度(从单体至五聚体)的片段被分开。胶经过NaOH和NaOAC处理,将DNA分子转移到DBM-滤纸上。此滤纸再经NaOH和H_2O处理,分别测定从单体至五聚体的不同分子量DNA的转移效率。结果指出,电泳胶经过转移前处理,单体和二聚体的损失分别为7%和5%,三聚体至五聚体没有损失。转移后仍留在电泳胶上的DNA为2—3%。经过转移后处理的DBM-滤纸所结合的DNA分子,从单体到五聚体分别为89%,91%,96%,94%和94%。因此,在所分析的分子量范围内,DNA分子基本上定量地被转移。用~(32)P标记的SV40 DNA作为探针,检测DNA碱变性与分子杂交效率的关系。结果指出,分子杂交之前,DBM-滤纸经NaOH的处理是必须的,但DNA被转移到DBM-滤纸之前,对电泳胶的碱处理步骤是可以省略的。     

Variation in mouse major urinary protein (MUP) genes and the MUP gene products within and between inbred lines

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.     

Bone morphogenetic protein activity preservation with extracorporeal irradiation- and liquid nitrogen freezing-treated recycled autografts for biological reconstruction in malignant bone tumor

Recycled autografts have been commonly used in biological reconstruction in conjunction with wide bone resection. Extracorporeal irradiation (ECIR) and freezing are the two major options for pretreating tumor-bearing autografts before transplant. This study, for the first time, compared the effects of these two techniques on bone morphogenetic protein (BMP)-2 activity. Bone tissue extracted from human femoral heads were treated through either ECIR at different doses (5000, 15,000, and 30,000 rad) or liquid nitrogen (LN) freezing for different durations (5, 10, and 15 min). The amount of BMP was analyzed through enzyme-linked immunosorbent assay (ELISA assay). Furthermore, we also used tandem mass spectrometry to analyze change of BMP-2 and BMP-7 expression after high dosage of irradiation (30,000 rad) and long-time of freezing (15 min). To directly evaluate the effect of ECIR or LN freezing treatment on the activity of BMP, commercial recombinant human BMP-2 (rhBMP-2) was added to the culture of human mesenchymal stem cells (hMSCs). The post-treatment activity of rhBMP-2 was quantitated by measuring the osteogenic differentiation of hMSCs with Alizarin Red S staining. Through Western blotting, the activation of the BMP signaling pathway by phospho-Smad antibodies was analyzed. Our results showed that post-treatment levels of BMP did not differ among the ECIR and LN freezing treatments in ELISA assay, but tandem mass spectrometry showed significantly lower expression of BMP-2 after 30,000 rad of irradiation. Both ECIR and freezing lowered the expression of regulatory factors involved in the BMP-activated signaling cascades and similar results were also observed in osteogenic differentiation of hMSCs. However, LN freezing preserved better bioactivity of rhBMP-2 whereas dosage-dependent declination was observed in ECIR groups. In conclusion, considering BMP-2 activity, LN freezing-treated autografts may result in a better osteoinduction outcomes than those treated using ECIR. Further investigation of the factors involved in bone formation is required.     

苦槛蓝挥发油对小菜蛾的生物活性

研究了苦槛蓝挥发油对小菜蛾的生物活性。结果表明,苦槛蓝挥发油对小菜蛾成虫具有强烈的驱避作用．小菜蛾成虫在四臂嗅觉仪中首选次数、平均停留时间从多到少的顺序是3臂、1臂、4臂、2臂,具有远离处理臂的趋势,其中雄虫比雌虫明显。且气体流速为200ml·min^-1时驱避作用最为显著。按方法3处理第一天对小菜蛾成虫的产卵驱避率(OCR)、干扰作用控制指数(IIPC)分别为94．48％、0．0552。此外,从挥发油中分离到一个液体成分,经鉴定确定为myoporone(C15H22O3)。     

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.