MECHANISM OF INTERFERON ACTION——STRUCTURAL REQUIREMENT OF LIGAND AND CELLULAR SPECIFICITY

We had reported the existence of 2'-5'P_3A_3 receptor on the peritoneal macrophages from Wistar rat. In this paper data are given to demonstrate that the ligand, for binding with 2'-5'P_3A_3 receptor and enhancing the phagocytosis of macrophages, must have a three-phosphate radical on its 5'-terminus and at least three adenylate residues in 2'-5'oligoadenylate. (~3H)-2'-5'P_3A_3 can also bind on macrophages from other animals; and the binding of (~3H)-2'-5'P_3A_3 on Hepatoma cell line 7402 is lower than that on macrophages; whereas it is very low (binding ratio less than 1%) on erythrocytes (from rabbit, rat and mouse) or human embryo lung fibroblast cells. The binding conditions of the ligand with 2'-5'P_3A_3 receptor have also been investigated.     

干扰素的功能表达机理——2′-5′三腺苷酸对巨噬细胞的激活对甲胎蛋白的对抗作用

本试验结果表明:1.2′-5′三腺苷酸(2′-5′P_3A_3)有激活多种来源(包括人)巨噬细胞的吞噬作用,说明2′-5′P_3A_3对巨噬细胞的激活作用具有普遍意义。2.甲胎蛋白(AFP)能抑制巨噬细胞的吞噬功能,可能对肝癌的发生有一定的作用。本工作证实2′-5′P_3A_3能抵消AFP对巨噬细胞的抑制作用。3.关于2′-5′P_3A_3生物功能的研究,过去都要用CaCl_2等方法将它引入细胞,这对临床应用是不实际的。本文不使用CaC国_2等方法,2′-5′P_3A_3即能激活巨噬细胞。     

酵母丙氨酸转移核糖核酸3′-半分子(36—76)的合成

本文报告用T_4RNA连接酶将相应于酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))3′-半分子(36—76)的三个寡核昔酸大片段——10(36—45)(Ⅰ),12(46—57)(Ⅱ)和19p(58—76)(Ⅲ)——从3′-端向5′-端延伸逐个连接合成了这个tRNA的3′-半分子(36—76)。首先在供受体配比为1:1.5的情况下,采取三步连续反应,即19p(Ⅲ)的5′-磷酸化,然后与12(Ⅱ)的连接和连接反应产物的5′-磷酸化等反应,一次制备分离的方法,以70％的总得率合成了5′-磷酸化的三十一核苷酸(46—76)(Ⅳ)。然后,以(Ⅳ)作为下一步反应的供体和三倍量的10(Ⅰ)连接,以67％的产率合成了具有四十一核苷酸的tRNA_y~(Ala)的3′-半分子(36—76)(Ⅴ)。将这个合成的3′-半分子,5′-磷酸化以后,与天然的5′-半分子连接,人工半合成了tRNA_y~(Ala)整分子,经生物活力测定,这个人工半合成的tRNA_y~(Ala)具有接受[~3H]-丙氨酸、并能将接受的丙氨酸转移到蛋白质分子中去的生物活力。     

MECHANISM OF INTERFERON ACTION——ACTIVATION OF MACROPHAGES AND ANTAGONISM TO a-FETO-PROTEIN BY _(ppp)A_(2'p5')A_(2'p5')A

_(ppp)A_(2'p),'A_(2'p,)'A(2'-5'P_3A_3) activates macrophages and increases the phagocytosis of macrophages from different species including human beings. This indicates that the activation of macrophages may be a general action of 2'-5'P_3A_3. This discovery broadens the effect of 2'-5'P_3A_3 beyond the antiviral field.α-Feto-protein (AFP) inhibits the phagocytosis of macrophages and may be involved in the development of hepatoma. Data presented here show that 2'-5'P_3A_3 can antagonize this suppressive effect of AFP.Methods used so far for introducing 2'-5'P_3A_3 into the cells were made with the aid of CaCl_2, etc. under conditions which may not be the same as those used clinically. It was found that 2'-5'P_3A_3 can develop its biological effect without the aid of CaCl_2, etc.     

MECHANISM OF INTERFERON ACTION——THE DISCOVERY OF pppA2'p5'A2'p5'A RECEPTOR

In this paper it is demonstrated that (~3H)2'-5'P_3A_3 can bind to the peritoneal macrophages from Wistar rat. This binding is strongly inhibited by cold 2'-5'P_3A_3 while the inhibiting capacity of 2'-5'P_3I_3 and 2'-5'A_3 is very small, showing that this binding is highly specific. And the binding of (~3H)2'-5'P_3A_3 is reversible, saturable and with high affinity. A Scatchard analysis of the binding data gives a linear plot, an apparent dissociation constant (Kd) about 1.3×10~(-7) M and binding sites per cell about 2.1×10~7. The above evidence shows the existence of the 2'-5'P_3A_3 receptor, a fact which, to our knowledge, has not been reported before.     

干扰素的功能表达机理——2′-5′三聚腺苷酸受体的发现

本文证明[~3H]-2′-5′P_3A_3能与Wistar大鼠腹腔巨噬细胞结合,这种结合能被冷2′-5′P_3A_3强烈抑制,而2′-5′P_3I_3及2′-5′A_3的抑制能力则很小,这说明结合的专一性较强;同时这种结合还具有可逆性、饱和性和高亲和性,经Scatchard作图法处理为直线关系,并算得Kd约1.3×10~(-7)M,每个细胞上约有2.1×10_(7)个结合部位。根据上述证据,说明巨噬细胞上存在2′-5′三聚腺苷酸的受体。这是2′-5′寡核苷酸类化合物受体的第一个报告。     

合成的人γ-干扰素cDNA在大肠杆菌的高效表达

合成了人γ-干扰素(IFN-γ)cDNA,其特点是选用了大肠杆菌偏爱密码子。将合成基因克隆到含P_L启动子的不同表达载体中,共获得9种不同的表达质粒,它们的SD与起始密码子ATG间的距离(D)不同,在起始翻译区(TIR)的最小自由生成能△G_(f298)~°不同,故其表达量的差异也很大。其中pLY_4-γ_5表达的IFN_γ的量占菌体总蛋白的60％—80％,为国际上所罕见。表达高的原因是由于选择(D)距离适当,△G_(f298)~°较高,以及选用的密码子较好。采用十分简便迅速纯化IFN-γ的方法分离包涵体可达90％以上纯度,再经一步分子筛柱层析,IFN-γ纯度即可达95％以上,比活约2.0×10~7IU/mg。     

2′-5′寡腺苷酸的有机合成与修饰

本文主要综述了三方面的内容:(1)2′-5′寡腺苷酸的生物学意义。(2)2′-5′寡腺苷酸的有机合成,特别是磷酸三酯法和铅离子(Pb~(++))催化法。(3)2′-5′寡腺苷酸结构与功能的关系,如5′端磷酸基,3′端羟基以及碱基与功能的关系。     

MECHANISM OF INTERFERON ACTION Ⅹ——ANTAGONISTIC EFFECT OF ATP ON pppA2''''p5''''A2''''p5''''A RECEPTOR

pppA2'p5'A2'p5'A (2'-5'P_3A_3) receptor may be the first one in the oligonucleotide field.But ATP can compete with 2'-5'P_3A_3 for the receptor. This raises a question whether this is anATP receptor. If not, what is the role of ATP for this receptor? Data available in this arti-cle show that ATP can bind to macrophages with saturability and reversibility. So ATPappears to be also a ligand of the receptor for 2'-5'P_2A_3. But the binding of ATP did notdevelop phagocytic effect as mentioned before~([11]). Moreover, ATP inhibited the enhancementof phagocytosis of macrophages by 2'-5'P_3A_3 when it acted together with 2'-5'P_3A_3 on mac-rophages at the same time. It is concluded that ATP is an antagonist of 2'-5'P_3A_3 receptor.When macrophages were treated with ATP prior to 2'-5'P_3A_3 and the binding sites for 2'-5'P_3A_3P were first occupied by ATP, both the binding ability and the biological effect of2'-5'P_3A_3 were all markedly blocked. The above data convincingly show that the action of2'-5'P_3A_3 is     

干扰素的功能表达机理——2′-5′三腺苷酸在干扰素抗病毒作用中的重要意义

本工作证实,pppA2′p5′A_2′p5′A(2′-5′P_3A_3)具有广泛的抗病毒作用,它能抑制RNA病毒如Influenza H_3N_2/77,Influenza H_1N_1/77,ECHO_(11),rhino,Sendal,Sindbis及VSV等病毒,也能抑制DNA病毒(如herpes Ⅰ型)在人胚皮肤肌肉成纤维母细胞中的致病,因而对病毒感染细胞起一定的保护作用。此外1μM2′-5′P_3A_3对个别病毒的作用效果(与其他病毒相比)甚至比512单位/毫升的干扰素(IFN)还好,这说明2′-5′P_3A_3在干扰素的抗病毒作用中具有很重要的意义。对2′-5′P_3A_3与IFN的抗病毒作用进行比较之后,发现对于多数病毒,二者虽有差异,但有一定的平行关系,少数病毒则二者差别甚远,所有这些差异可能是由于病毒本身的作用所引起。