Prion diseases (transmissible spongiform encephalopathies, TSE), as a group of fatal neurodegenerative diseases, have affected humans and a variety of other mammals. Although no natural TSE have been documented in pigs, appropriate precautions need to be taken to prevent the iatrogenic spread of prion disease through pig-to-human xenotransplantation. Polymorphisms within the open reading frame (ORF) of the single-copy gene of prion protein (PRNP) are associated with susceptibility to scrapie in sheep and variant Creutzfeldt-Jacob disease in humans. We screened polymorphisms in the PRNP gene of 64 China Experimental Minipigs and Beijing Large White pigs. Our findings suggest that the porcine PRNP gene is highly homogenous. The amino acid sequences of the mature prion protein of all samples tested were identical. Four single nucleotide polymorphisms (G11A, G615C, G684A, T726G) in the ORF of the porcine PRNP gene were found, and the G fi C nucleotide substitution resulted in a serine to asparaginate amino acid substitution at codon 4. We conclude that pigs raised under specific pathogen-free conditions, with the exclusion of rendered mammalian material for at least two generations, will have little risk of being infected with a TSE, and even less possibility of transmitting prion disease to humans through xenotransplantation.

A Yunnan pony ear marginal tissue fibroblast cell line (NYPEM 2/2) was successfully established using the ex2 plant of the ear marginal tissue and then trypsinization the cells from the outgrowth. Observations on cell morphology and dynamic growth, analysis of karyotype and isoenzymes of lactate dehydrogenase and malate dehydrogenase were carried out. The expression of recombinant green fluorescence protein in the cells were also undertaken. The results showed that the population doubling time (PDT) of the cells was 24h; the frequency of cell chromosome number to be 2n=64 was 9219%; the banding patterns of the isozymes of the two enzymes had significant difference between the Yunnan pony ear marginal fibroblast cell line and the fibroblast cell lines of PEM 2/2, MSHEM 2/2 and BLCHE 2/2 derived from the Picdmont bovine ear, Mongolian ovine ear and Beijing local chicken embryo respectively. Tests for the contamination from bacteria, fungi or mycoplasma were negative; the transfection efficiency for the recombinant plasmid was 3213%. This newly established cell line make the Yunnan pony breed, a national important genetic resource preserved at cell level, as well as will provide an effective experimental material for genetic studies on the Yunnan pony

Background: After menopause women are more susceptible to coronary heart disease due to increased risk of atherosclerosis. Tibolone (Livial) is an innovative synthetic steroid analogue for the treatment of postmenopausal climacteric symptoms including atherosclerosis, but the mechanisms of its effect are still unclear. The present study investigated the effect of tibolone and simvastatin on atherosclerosis and the expression of both estrogen receptor A (ERA) and LDL receptor (LDLR) mRNA in ovariectomized cholesterol-fed rabbits. Methods: Fifty New Zealand white rabbits were included for the study. Of them, 40 underwent bilateral ovariectomy and the other 10 were sham-operated. The sham-operated group only received atherogenic diet (group SC) and the ovariectomized rabbits were divided into 4 groups of 10 each, with group N received normal diet, group C received atherogenic diet, group T received atherogenic diet and tibolone (2.5mg/day) and group SI received atherogenic diet and simvastatin (20mg/day). After 12 weeks of the treatments, the animals were euthanized and the extent of thoracic aortic atherosclerosis was measured morphologically and the level of ERA and LDLR mRNA in heart and liver was determined by real-time RT-PCR. Results: The extent of atherosclerosis in the thoracic aorta was 0.75

The Debao pony ear marginal tissue fibroblast cell line (NDPEM 2/2) was successfully established using either primary explant technique or collagenase technique. The characterizations of the cell line were identified as following: the cells were adherent and of density limitation; population doubling time (PDT) of cells made with the two techniques were 35.9h and 48h, respectively; chromosome analysis showed that the frequency of cell chromosome number to be 2n=64 was 91.3%-92.8%. Confirmed by isoenzyme analysis, this cell line had no cross- contamination. Tests for microbial contamination from bacteria, fungi, virus or mycoplasma were negative. This newly established cell line meets all the standard quality controls of ATCC. It will provide a precious genetic resource for the conservation of the Debao pony breed, as well as effective experimental material for genetic studies on Debao ponies.

The open reading frame of peacock and parakeet prion protein (PrP) genes was cloned and sequenced. The peacock and parakeet PrP genes consisted of 833 and 866 nucleotides encoding 266 and 277 amino acids, respectively (GenBank Accession numbers AY365065 and AY365066). Sequence analysis showed that the peacock and parakeet PrP genes had 93.67% homology to each other, 94.04% and 99.64% homology to the chicken PrP gene and 46.0% and 42.1% similarity to the mammalian PrP genes, respectively. The structural features of all known mammalian and avian PrPs, including N-terminal signal peptides, tandem repeats, conserved hydrophobic region, disulfide bridges and glycoinositol phospholipids anchor, were also found in peacock and parakeet PrPs. The parakeet and peacock PrPs, however, differed in the hexarepeat region, with the peacock having six and the parakeet having seven hexarepeats. The phylogenetic analysis showed that the PrP genes in 52 species were clustered into 2 distinct lineages, the avian and the mammalian. The peacock and parakeet PrP genes belonged to the same lineage but the peacock PrP was sub-classed with the pigeon PrP and the parakeet PrP was sub-classed with the duck and chicken PrPs. The present work added two more species data to the collection of the PrP genes and supported the previous findings that the PrP genes are highly conserved across species.

Determination of tissue-specific expression of cellular prion protein (prpc) is essential for understanding its poorly explained role in organisms. Herein we report on quantification of prp Mrna in golden hamsters,a popular experimental model for studying mechanisms of transmissible spongiform encephalopahies (TSE), by real-time RT-PCR.Total RNA was isolated from four different regions of the brain and six peripheral organs of eight golden hamsters Prp Mrna copy number were determined using absolute standard curve method with real-time quantitative PCR instrument.I was found that high Mrna levels were present in all four regions of the brain examined,ingunal lymph node showed high level of the expression similar to that in overall brain; spleen, heart, liver, and lung showed moder-ate levels of the expression; and kidney showed the lowest expression. Our result is consist-ent with the potential involveoment of different organs in prion diseases and offers essentialdata for further study of TSE mechanism in this animalmodel.

根据已报道的哺乳动物朊病毒基因序列设计引物对，采用PCR方法扩增了大熊猫的朊病毒基因，将其克隆到T2Easy载体，序列测定及分析表明所克隆的大熊猫PRNP基因（GeneBank收录号为AF327449）片段为795bp，编码264个氨基酸的前体蛋白，推测其分子量约28.5ku。与已报道的牛（GeneBank收录号为AF455119）、绵羊（GeneBank 收录号为AF367623）的相应序列作比较分析，核苷酸序列同源性分别为99%和83%，其编码的氨基酸同源性均为100%。在所克隆的大熊猫PRNP基因中未发现与朊病毒敏感性连锁的氨基酸多态性位点。

以朊蛋白单抗AH6和碱性磷酸酶标记的酶标马抗鼠二抗建立了疯牛病和羊痒病的Western Blot检测方法。对Western Blot各种反应条件进行摸索，并确定了最佳工作条件，结果表明：当匀浆缓冲液为RIPA时最佳反应条件为浓缩胶内电泳电压为恒压90V，分离胶电泳电压为恒压160V，转印的最佳电压和时间为恒压100V 1.5小时；封闭液为3%BSA时，封闭15分钟，封闭效果最好；AH6的最佳稀释浓度为1：4000，4℃下孵育过夜，马抗鼠二抗的最佳稀释浓度1：1000，室温下孵育30 分钟。采用已确立的反应条件对样品进行检测并与Prionics®-Check WESTERN进口试剂盒的检测结果比较，发现其敏感性为100%，特异性为99.4%，而进口试剂盒分别为100%，100%，与进口试剂盒无显著差异，这为在该基础上建立国产试剂盒提供了条件

金黄地鼠是研究动物传染性海绵状脑病的理想模型动物之一，我们利用实时荧光定量RT-PCR技术，构建标准重组质粒制备标准曲线，对中枢神经系统的4个不同部位及外周6个组织提取总RNA，反转录后进行PrP基因表达的定量。结果发现，脑的四个检测部位都呈现高的表达量；在外周器官中，淋巴结的表达量和全脑相当，脾脏、心脏、肝脏和肺脏呈现中等程度的表达，肾脏的表达量最低。本研究的结果对于探讨朊蛋白的基本功能和不同组织在传染性海绵状病理发生中的作用，提供了基础数据。