三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

关于茭白黑粉菌的正名问题

茭白黑粉菌在文献中出现Yenia esculenta(P.Henn.)Liou及Ustilago esculenta P.Henn.两个名字。现经形态发生学的研究以明确新名字是否有效。此菌冬孢子没有休眠期,孢子成熟后在适宜的环境中即可萌发。萌发的最适pH值为6;最适温度为25％;不需要外界光的诱导,在黑暗条件下也能正常萌发;培养基中的营养成份对孢子萌发的速率有一定影响。冬孢子在不同的环境条件下,其萌发形态及方式都是稳定的:孢子萌发时产生有隔的担子或先菌丝(promycelium)。该先菌丝起初可以是无隔的,但生长到一定程度,便产生分隔,分隔多为2—4个。初生的先菌丝和小孢子是单核的,但成熟的先菌丝、小孢子及短菌丝细胞内均是双核的。可以认为,该菌的双核期在整个生活史中占有相当长的时间,它只有短暂的单核期。没有发现先菌丝、小孢子彼此之间或相互间融合的现象;未见到双核的融合及减数分裂过程。茭白黑粉菌在人工组合培养基上生成厚壁的孢子,较自然界中的冬孢子为大,但萌发方式则相同。最后作者等认为茭白黑粉菌仍应保留在Ustilago属中,而Yenia esculenta(P.Henn.)Liou的名字是无效的。     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Concerted generation of Ig isotype diversity in human fetal bone marrow

The human fetal bone marrow B cell compartment of 14- to 21-wk gestational age was examined phenotypically and with respect to Ig H chain commitment and diversity. A dramatic expansion of fetal marrow B cell pools at 16- to 18-wk gestational age characterizes a rapid and concerted chain of differentiation events. Transiently up to 1/4 of nucleated marrow cells are CD20+/CD21+ cells which begin to express surface Ig other than IgM. Limiting dilution analysis of EBV-infected marrow cells delineated a virtually exclusive commitment to IgM production until 15 wk and the absolute and relative number of these cells were small (approximately 5% of comparable adult values). In parallel to the rapid increase in total B cell pools size, cells committed and able to secrete any of the five Ig isotypes are generated by 16-wk gestational age and by 18 wk the frequencies of these cells rapidly reach levels typical for adult peripheral tissue such as blood or lymph node. Fetal L chain diversity always anticipated that observed in adult serum. In addition to rising pool sizes and diverse IgH expression, EBV transformability is a major variable during this period of B cell development with up to 2/3 of B lineage cells transformable, about half of which are pre-B cells. By 21-wk gestational age transformable pre-B cells have disappeared and (as in adult tissue) approximately 10 to 20% of CD20+ cells are transformable. The rapid, concerted expression of full H chain diversity during a narrow period in fetal development is unique to marrow and implies a lymphopoietic process in a privileged site rather than an immunologic differentiation event. During this event, the relative proportions between the different IgH classes expressed, resembled that found in adult tissue, perhaps suggesting that B cell inherent programming rather than only antigenic forces determine heavy chain choice. The staggered expression, early in postnatal life, of IgH regions 3' of the C mu locus may reflect regulatory functions rather than inherent immaturity of the B lineage.     

Minimal sequence requirement of thrombin receptor agonist peptide.

A site-specific proteolytically generated neoamino terminus of the thrombin receptor having a sequence SFLLRNPNDKYEPF- has been reported to be a functional ligand of the receptor. This discovery raises question on the precise structural requirements of the "tethered ligand" responsible for receptor activation and signal transduction. By examining the agonist activity of a panel of synthetic sequence analogues of thrombin receptor agonist peptides (TRAP) on human platelet aggregation, we determined that the minimal sequence of the human platelet thrombin receptor ligand is SFLL-amide (TRAP1-4, EC50 = 300 uM). An extension of TRAP1-4 by an additional Arg-Asn segment yielded the most potent agonist among the series (TRAP1-6, EC50 = 1.3 microM). Based on the structure-activity relationships, we hypothesized a model of the ligand-binding site of the human platelet thrombin receptor that accommodates a hexapeptide structure. TRAP1-6, when administered intravenously, induced marked intravascular platelet aggregation in the anesthetized guinea pigs.     

家兔脊髓蛛网膜下腔注射乙酰胆碱对血压和心率的影响

将乙酰胆碱(ACh)注入麻醉家兔脊髓蛛网膜下腔,观察其对心血管活动的影响。结果表明:(1)脊髓蛛网膜下腔注射50～100μg ACh可使血压下降,心率减慢;(2)预先由脊髓蛛网膜下腔注射阿托品,可阻断ACh引起的降压和降心率作用;(3)脊髓蛛网膜下腔注射六甲双铵、酚妥拉明或心得安均不能阻断上述ACh的心血管反应;(4)切断两侧颈部迷走神经,ACh不再使心率减慢,但其降低血压的作用不受到任何影响。 脊髓中ACh水平升高可通过激活胆碱能M-受体引起血压下降和心率减慢。ACh的这种降压作用既没有中枢肾上腺素能受体活动参与,也不是通过迷走神经实现的,可能是由于脊髓交感血管中枢紧张性降低所造成的。     

电针引起脊髓P物质释放的频率依赖性

我室以往的研究表明,不同频率的电针可引起脊髓释放不同种类的阿片肽。本工作观察Ｐ物质（ＳＰ）释放的频率依赖性,电针频率选择２,４,８,１５,３０和１００Ｈｚ,分别收集电针期间及电针前后各３０ｍｉｎ的脊髓灌流液,通过放射免疫方法测定大鼠电针有效组和电针无效组Ｐ物质免疫活性（ＳＰ－ｉｒ）．结果如下：（１）电针有效组：２Ｈｚ引起ＳＰ－ｉｒ降低,与电针前相比,Ｐ＜０．０１;４Ｈｚ电针前后ＳＰ－ｉｒ比较,无统计学意义;８,１５,３０,１００Ｈｚ时ＳＰ－ｉｒ均增加（Ｐ＜０．０１）,其中１５Ｈｚ时ＳＰ增加最多（Ｐ＜０．００１）,表明刺激引起ＳＰ释放有频率依赖性。（２）电针无效组：不论应用何种频率,电针前后脊髓灌流液中ＳＰ－ｉｒ变化不大（均Ｐ＞０．０５）。提示,电针时脊髓液中ＳＰ含量变化与镇痛效果有密切关系。     

小麦×玉米杂交后代的蛋白质及酯酶同工酶分析

以８ 个普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）品种为母本,２ 个栽培玉米（Ｚｅａ ｍ ａｙｓ Ｌ．）品种为父本杂交所获得的Ｆ２ 代在形态上出现了明显变异。对其籽粒进行蛋白质电泳分析,得到了如下主要结果：杂交后代的蛋白质谱带较母本有了很大的变异,主要集中在高分子量麦谷蛋白（ＨＭＷ－Ｇｌｕ）区域。杂交后代的蛋白质谱带由５ 种类型构成：１．母本型,占全部测试籽粒的２２．６％ ;２．附加型,占１４．３％ ;３．互补型,占１５．５％ ;４．杂种型,占３０．９％ ;５．缺失型,占１６．７％ 。对“矮杆早”ד紫粘”的Ｆ２ 代籽粒进行酯酶同工酶电泳分析发现,变异主要发生在ＥＳＴ－１ 区。由此看来,小麦×玉米的杂合子中玉米染色体在被排除前后,可以诱发小麦染色体组发生遗传变异     

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl₂ at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys¹⁴ to Cys⁷⁹, Cys⁵⁷ to Cys¹⁰⁸, and Cys⁶⁷ to Cys¹¹⁰. This disulfide structure is homologous to the NGF family of neurotrophic factors.     

细小病毒非结构蛋白转基因小鼠模型建立

将小鼠乳腺癌病毒启动子控制的细小病毒非结构蛋白基因(长5.7kb)氯化铯超速离心,纯化透析后用显微注射法导入C57BL/SJL F_1小鼠受精卵雄核,植入假孕母鼠输卵管,得成活小鼠15只。抽取鼠尾DNA,对其中10只小鼠作PCR和southern blot鉴定,其中4只(40％)整合有目的基因。对首建者B_6()的8只子代小鼠鉴定,3只(37.5％)整合有目的基因。说明导入的目的基因能传代。