我国十省(市)十字花科蔬菜芜菁花叶病毒(TuMV)株系分化研究 Ⅰ.用Green氏方法划分株系

Provvidenti,R.(1980)和Green,S.K.(1985)先后用同一套鉴别寄主谱,研究了美国和我国台湾省的TuMV株系分化情况。本文用Green的方法,对由十省(市)7,982份病样中筛选出的19个TuMV主流分离物,在同一条件下进行了鉴定。结果表明,属于C_1株系的有黑_3分离物;属于C_4株系的有京_2、京_3、冀_2、宁_1(南京)、粤_1和川_1共6个分离物;属于C_5株系的有黑_1、辽_1、京_1、冀_1、沪_1、鲁_1、鲁_2共7个分离物。未检出C_2和C_3株系。黑_2、沪_2、宁_2和秦_1(陕西)共4个性状相近的分离物和冀_3分离物尚不能按Green的标准归类,暂分别定为C_(3-2)和C_6株系。此外,对Green氏方法应用于我国十字花科蔬菜TuMV株系分化研究中存在的问题作了讨论。     

皮层第二体感区下行活动对尾核神经元伤害性反应的影响

本实验用清醒麻痹的家猫观察到,皮层第二体感区(SⅡ区)的下行活动对尾核痛相关单位的自发放电和伤害性反应均有影响。在35个痛相关单位中,刺激SⅡ区后,62.7％的自发放电受到影响(p<0.05)。在72个痛相关单位中,刺激SⅡ区后,77.8％的伤害性反应受到抑制(p<0.01),其中抑制痛兴奋单位的伤害性反应最为明显。而用利多卡因局部阻滞SⅡ区后,33个痛相关单位中,51.5％的伤害性反应增强(p<0.01)。本结果提示:(1)SⅡ区的下行活动不仅对尾核痛相关单位的自发活动有影响,而且对痛相关单位的伤害性反应有以抑制为主的下行性调制作用。(2)在正常机能状态下,SⅡ区对伤害性信息向尾核的传递有一定的紧张性的下行性抑制作用。     

白细胞介素2受体α链基因转录起始点和5'调控区结合蛋白的初步研究

 借助于5'和3'末端删切后重建的IL-2R a链基因调控区次级克隆,在体外合成有放射性同位素参入的反意义RNA探针与总RNA进行液相杂交,结果表明TPA或PHA分别活化的T细胞在IL-2R a链表达过程中都在不同程度上有选择地利用了调控区内分别为-58(5')和+1(3')位两个转录起始点中3'转录起始点。热休克使PHA活化细胞更明显地利用+1位点。PHA诱导Jurkat细胞表达IL-2RamRNA斑点杂交证实,Jurkat细胞在活化16小时表达IL-2Ra基本达到高峰,至24小时已明显下降。根据这一规律提取PHA诱导活化15小时的Jurkat细胞S100和NE,进行有关结合蛋白的研究,初步结果显示磷酸纤维素柱的KCI洗脱组分中存在着DNA结合蛋白,有关结合蛋白性质的研究正在进行中。     

用高碘酸盐活化葡聚糖凝胶制备亲和吸附剂的研究

 <正> 用高碘酸盐活化的Sephadex与鸡卵类粘蛋白偶合制备分离纯化胰蛋白酶的亲和吸附剂。该方法比CNBr活化Sepharose 4B制备亲和吸附剂的方法具有操作安全,价格低廉等优点。活化载体在4℃保存较长时间不失去键合能力。该亲和吸附剂可制备得比活力为11228.8u/mgBAEE单位电泳单带纯胰蛋白酶,并能反复使用十余次,仍具有较强亲和吸附能力。酶     

用定点突变技术研究转录终止——rpoBC操纵子上~tL7茎环结构的作用研究

 E.coli RNA聚合酶ββ'亚单位编码的基因rpoBC与核糖核蛋白基因rplJL共同构成rpoBC操纵子。rpl-rpo间区转录终止信号~tL7的转录产物RNA有两组富含C-G的反向对称结构及一串寡聚U;反向对称区可形成1:2和3:4茎环或单一5:6茎环。 用M13mp11噬菌体插入~tL7的112bp片段重组M13mp11-490,在此基础上用定点突变技术建立~tL7的七核苷酸缺失突变体,从而破坏1:2茎环,建成M13mp11-85。 分别把原型~tL7及缺失型~tL7~v克隆到表达质粒pHR24中,构建成pHR37(P_ftsQ~tL7-galk)及pHR24-9(p_ftsQ-~tL7~v-galk)。测定galk基因产物半乳糖激酶活性,推算转录的终止效率。结果表明:~tL7终止效率为50％,~tL7~v为20％。说明仅有3:4茎环及寡聚U,即具终止作用; 1:2茎环通过某种方式加强3:4茎环从而提高终止效率。     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.     

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.