鱼类HGPRT缺陷型细胞的诱变和筛选

草鱼卵巢细胞ＧＣＧ经乙基甲磺酸（ＥＭＳ）诱变，稳定表达３代以后，采取逐步提高培养基中６－疏基嘌呤（６－ＭＰ）的方法，获得对６－ＭＰ稳定抗性的细胞，暂定名ＦＭＲ－１．本文比较了ＧＣＧ细胞及ＦＭＲ－１细胞在含６－ＭＰ（２０μｇ／ｍｌ）和不含６－ＭＰ的培养基中生长曲线的特点，并对上述两种细胞的染色体数目和葡萄糖－６－磷酸脱氢酶电泳图谱进行了比较分析。最后对两种细胞在ＨＡＴ培养基中的生长特点进行了平行对照实验，根据实验结果，可以判定ＦＭＲ－１细胞为草鱼ＨＧＰＲＴ缺陷型细胞。本义还讨论了进一步研究草鱼ＨＧＰＲＴ缺陷

Cytotoxicity, genotoxicity and antimutagenicity of hexane extracts of Agaricus blazei determined in vitro by the comet assay and CHO/HGPRT gene mutation assay

Agaricus blazei Murrill ss. Heinem, known as the sun mushroom or himematsutake, is a basidiomycete native to Brazil, which is popular for its medicinal properties. The aim of this study was to test hexane extracts (one fraction and its four sub-fractions) of A. blazei for bioactivity in cultured mammalian cells (CHO-K1). The comet assay, the colony forming assay (CFA) and CHO/HGPRT gene mutation assay were used respectively to determine genotoxicity, cytotoxicity and antimutagenicity of these extracts at different concentrations. The cells were incubated in culture medium and treated for 3 h according to the standard protocol for each assay. The DNA damage-inducing agent ethylmethane sulfonate (EMS) was utilized as the positive control and also in combination with extracts to test for a protective effect. Statistical analysis of the data was performed using analysis of variance (ANOVA) and Tukey's test. A relationship between cytotoxicity and genotoxicity could be established and two extracts EH6B and EH6D showed a protective tendency, while the others did not, with the primary extract EH6 causing the most substantial damage to genetic material. These findings warrant more in-depth studies of the active principles of this mushroom. (c) 2005 Elsevier Ltd. All rights reserved.

麻疹疫苗诱发中国仓鼠卵巢细胞(CHO-K_(1))染色体畸变和基因突变的研 究

以中国仓鼠卵巢细胞(CHO)次黄嘌呤-乌嘌呤磷酸核糖转移酶位点突变检测系统(CHO/HGPRT)检测国产麻疹疫苗的诱变性。实验证明,麻疹病毒及其疫苗可诱发染色体畸变。接种的三个剂量, 对细胞存活率无显著影响(P>0.05),但能使HGPRT位点正向突变频率升高。2.8logTC1D_(50)/ml和3.1logTC1D_(50)/ml组的突变频率的升高与对照组相比有统计学意义。表明HGPRT~(-)突变频率的升高与接种疫苗有关。

麻疹疫苗诱发离体中国仓鼠卵巢细胞染色体畸变和基因突变的研究

CHO／HGPRT系统测定国产疹活疫苗的诱变活性。结果表明：（1）麻疹疫苗可诱发中国仓鼠卵巢细胞（CHO）次黄嘌呤－－鸟嘌呤磷酸核糖转移酶（HGPRT）位点正向突变；接种量为3.8logTCID_(50)／瓶时和4.1logTCID_(50)/瓶时，诱发突变频率的升有统计学的意义；（2）麻疹疫苗对CHO细基本不表现杀细胞的毒性；相反，还有提高细胞克隆形成率的趋势；（3）在我们的实验条件下，麻疹疫苗诱发的染色体断率和多倍体率无明显的变化。上述结果提示：由麻疹病毒减毒株制备的麻疹活疫苗，在基因水平上，具有在的诱变活性；但在染色体水平上，未见明显的诱发断裂的活力。

Hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv: Cloning, expression, and biochemical characterization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

Lead compounds for antimalarial chemotherapy: Purine base analogs discriminate between human and P-falciparum 6-oxopurine phosphoribosyltransferases

The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K-m values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50-80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC50 values as low as 1 mu M.