1,4—二取代酰氨基硫脲类化合物(DATCD_s)的植物生理效应与应用研究——Ⅵ.DATCD—A对离体黄瓜子叶中RNA代谢的调控

DATCD—A抑制RNA水解酶活性,提高RNA合成速率,诱导离体黄瓜子叶中与RNA基因达相关的poly(A)RNA含量的增加,并增加总RNA的含量。     

不同降温模式对天然气水合物合成的影响

水合物实验室合成过程中,降温模式对诱导、结晶阶段都具有明显的影响。反应温度过低时,表现为反应速率慢、诱导期长,同时温度过高时,随着反应的进行压力降低,生成水合物会分解。基于升温提高合成反应速率的同时,为了形成稳定、均一分布的高质量水合物样品,在一次降温的基础上,进行了4～2℃和3～1℃两种阶梯降温和4轮次温度震荡的不同降温模式实验研究。结果表明:阶梯降温为3～1℃时比4～2℃诱导期略有增加,但最终形成时间相差不大,同时压降明显增大,气体转化率提高;增加温度震荡次数,压降逐渐增大,但具有次效性。可见阶梯降温和温度震荡均有利于水合物的增量合成,形成多孔介质中分布均一的水合物。     

抗氧化剂Isoverbascoside对MGC80-3细胞抗氧化酶活性和癌基因表达的影响

以苔酚蓝拒染法、核黄素-NBT还原法、DTNB还原法及RNA斑点杂交分析法分别对经不同浓度Isoverbascoside(Isov)处理的MGC80-3 细胞的生长速率、II期抗氧化酶(超氧化物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶)活性和N-ras、C-m yc及p53 基因表达作了检测.结果显示:经Isov处理后,细胞生长明显受抑;II期抗氧化酶活性较处理前明显上升;癌基因N-ras、C-m yc的表达较处理前下降,而抑癌基因p53 表达上升. 表明Isov 的抑癌作用与诱导II期抗氧化酶活性升高和癌基因表达改变有关     

CO_2水合物生成的影响因素研究

在体积350 m L的恒容反应釜中研究了CO2水合物的生成过程,实验考察了搅拌速率、温度和压力等因素对CO2水合物生成过程的影响。实验结果表明,相对静止条件下机械搅拌可以通过促进传质传热,加速CO2水合物生成过程。随着搅拌速率的增大,CO2水合物的平均生长速率从0.283 mmol/min增大到1.132 mmol/min,且搅拌速率为800 rpm时实验条件下的水合物的诱导期最短;不同实验温度(273.65 K,274.15 K,275.15 K,276.15 K)下,CO2水合物生成过程中的溶解速率、诱导期及反应速率变化不大,即受温度影响不明显;CO2水合物的生长受压力影响明显,增压可显著缩短诱导期、加快反应速率。水合物生长过程主要受客体分子浓度的影响,是动力学控制过程。3 MPa时CO2水合物的平均生长速率高达1.1 mmol/min,是2 MPa时的5倍。     

TRPV1在LPS引起的大鼠发热伴疼痛或痛觉过敏中的作用

为探讨TRPV1在LPS引起的大鼠发热伴疼痛或痛觉过敏中的作用。采用经大鼠侧脑室给予TRPV1受体阻断剂辣椒平(Capsazepine,CPZ)、30 min后腹腔给予LPS诱导发热模型的方法。通过微电脑测温仪和足底疼痛测试仪间断观察大鼠体温(Tc)及痛觉潜伏期的变化;实时荧光定量PCR(RT-q PCR)检测给予LPS 6h大鼠POA和L3-L5段DRG部位TRPV1m RNA表达变化。结果显示:与对照组相比,CPZ组Tc和痛觉潜伏期无明显变化;LPS组Tc明显升高,潜伏期明显缩短;CPZ+LPS组Tc显著升高,但潜伏期明显延长。与LPS相比,CPZ+LPS组Tc升高显著,持续时间更久,潜伏期延长更加明显。相比Control组和CPZ组,LPS组POA和DRG部位TRPV1m RNA相对表达量均显著增加,CPZ+LPS组TRPV1m RNA相对表达量在POA部位无明显变化,在DRG部位显著增加。由此可知,正常体温状态下,TRPV1未参与大鼠体温及痛觉过敏的调节;通过阻断TRPV1使发热大鼠体温进一步升高,发热时程延长,痛觉潜伏期延长,表明发热时TRPV1被激活,对LPS诱导的发热具有一定的负调控作用,同时提高了痛觉敏感性;TRPV1可能在外周和中枢共同参与了体温调节和痛觉调制。     

抗菌肽SMAP-29在毕赤酵母中的表达

依据毕赤酵母密码子偏好性,设计合成抗菌肽SMAP-29成熟肽基因片段,克隆到表达载体pPIC3.5K上,SalI线性化后转化毕赤酵母GS115,418抗性筛选高拷贝克隆,再由酵母菌落PCR鉴定;阳性克隆用甲醇诱导表达,Tricine-SDS-PAGE分析,结果在诱导第2d的酵母裂解液中检测到与预期的SMAP-29分子量接近,约3.2kD的诱导表达带;Trizol法提取酵母总RNA,并通过RT-PCR扩增SMAP-29mRNA,发现表达期的酵母细胞中存在SMAP-29mRNA,而对照没有检出,表明SMAP-29在毕赤酵母中存在表达。     

关于在尾穗苋黄化幼苗中影响苋红素合成的几个因素的分析

不同苗龄的尾穗苋黄化苗,光照诱导苋红素合成的效应,以在激动素溶液中为最强,酪氨酸中次之,而以在水中为最弱。苋红素的合成,不但对光照存在敏感期,对激动素也同样存在敏感期。但后者持续时间较长。黄化苗根部对苋红素合成有促进作用。光照强度在诱导苋红素合成方面,无明显作用。光照时间对苋红素合成的影响与幼苗本身对光照的敏感性相关。     

电化学调控ATRP动力学模拟的初步研究

基于矩方法建立了电化学调控下的原子转移自由基聚合（electrochemically mediated Atom transfer radical polymerization,eATRP）的动力学模型。利用上述模型,分析了eATRP与普通ATRP之间区别,并预测了过电位、催化剂浓度和引发剂浓度对聚合反应的影响。模拟结果表明,过电位的增大对聚合反应速率促进明显,但聚合物分散指数不变;增大催化剂浓度可以提高聚合反应速率,同时降低PDI。当催化剂浓度低至30ppm时,聚合物分散指数(polydispersity index, PDI)在反应时间内不能达到稳定且高达1.2以上;引发剂的影响主要体现在反应的诱导期长短上,当引发剂与单体浓度比下降至0.5:300时,诱导期从200s迅速增大到400s左右。     

电化学调控原子转移自由基聚合动力学模拟初步研究

基于矩方法建立了电化学调控原子转移自由基聚合(eATRP)的动力学模型.利用该模型分析了eATRP与普通ATRP之间的区别,并模拟了过电位、催化剂浓度和引发剂浓度对聚合反应的影响.模拟结果表明,过电位的增大对聚合反应速率的促进作用明显,但聚合物分散指数(PDI)不变;增大催化剂浓度可以提高聚合反应速率,同时降低PDI.当催化剂含量(催化剂与单体浓度之比)低至3×10-5时,PDI在反应时间内不能达到稳定且其值高达1.2以上;引发剂的影响主要体现在反应的诱导期长短上,当引发剂与单体浓度比下降至0.5…300时,诱导期从200s迅速增大到400s左右.     

青霉菌（Penicilliumsp.91—4）合成菊粉酶的调节

探讨青霉菌（Ｐｅｎｉｃｉｌｉｕｍｓｐ．９１－４）合成菊粉酶的调节控制时发现该菌株菊粉酶合成速率与菌体生长速率呈负相关，酶的合成受菊粉诱导和菊粉降解物阻遏调控．通过放线菌素Ｄ等抑制剂对酶合成影响的研究，认为菊粉酶生物合成时降解物的阻遏发生在转录水平．阻遏条件下菌体ＡＴＰ水平比诱导条件下高１０３倍，菌体ＡＴＰ水平是反映青霉９１－４菊粉酶合成状况的重要指征     

实验性“脾虚”动物胰腺细胞代谢及功能变化的研究

用西宁西大黄水浸煎剂使雄性金黄地鼠泄泻致虚,再用“四君子汤”复健。注射[5-~3H]尿嘧啶核苷或D.L(4,5)~3H亮氨酸分别研究核酸或蛋白的合成功能。表明脾虚动物胰腺合成蛋白质和分泌消化酶功能的下降与脾虚症状的发展相关。“四君子汤”对其有显著改善作用。     

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.     

Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.     

Does Q beta replicase synthesize RNA in the absence of template?

Q beta replicase, in the absence of added template, will synthesize RNA autocatalytically. A variety of small RNa species, termed '6S RNAs' are generated. As this reaction purportedly occurs in the absence of template, it has been termed 'de novo' RNA synthesis. The question of whether Q beta replicase can polymerize replicatable RNA molecules, without instruction from a template, has important evolutionary implications. The finding that Q beta replicase was able to synthesize RNA de novo was based on (1) failure to find contaminating RNA in Q beta replicase preparations; (2) differences in the sizes of products of apparently identical reactions; and (3) kinetic differences between template-instructed and de novo reactions. Here wer describe a procedure for production of Q beta replicase lacking one of its subunits, ribosomal protein S1, involving column chromatography in the presence of a low concentration of urea. We show that the resulting highly purified enzyme will not synthesize detectable RNA in the absence of added template. We show also that the ability to perform a reaction kinetically indistinguishable from the de novo synthesis reaction can be restored to the highly purified enzyme by adding a heat-stable, alkali-labile component of Q beta replicase preparations. Thus our findings suggest that, in the novo reaction, Q beta replicase is replicating previously undetected contaminating RNA molecules.     

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).     

Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase

The initiation of protein synthesis can be regulated in mammalian cells by protein kinases which phosphorylate the alpha subunit of initiation factor eIF-2. This phosphorylation results in a block in the recycling of eIF-2 and in the inhibition of messenger RNA binding to 80S initiation complexes. After eIF-2 alpha is phosphorylated, the mRNA becomes associated with 48S complexes consisting of a 40S ribosomal subunit, eIF-2 (alpha P), GDP and Met-tRNAf. One of the eIF-2 alpha kinases is activated by low concentrations of double-stranded RNA (dsRNA). This kinase (PKds) is present at a basal level in all mammalian cells investigated and its synthesis is induced in cells treated with interferon. The PKds may be involved in the inhibition of translation of viral mRNA in interferon-treated cells infected with RNA viruses, as it is activated by viral replicative complexes. It is not known, however, if the activated PKds preferentially inhibits the translation of viral mRNA when cellular protein synthesis proceeds at a normal rate in infected cells. We now report that mRNA covalently linked to dsRNA is preferentially inhibited from binding to 80S complexes by a localized activation of PKds. This suggests that in interferon-treated cells the binding of some nascent viral mRNAs to functional initiation complexes may be preferentially inhibited by a similar mechanism.     

2'5' oligo(A) polymerase activity in serum of mice infected with EMC virus or treated with interferon

Interferon-treated cells show an increase in two double-stranded RNA (dsRNA)-dependent enzymatic activities involving an oligoadenylate polymerase and a protein kinase (ref. 1 and refs therein). The polymerase converts ATP into a series of oligonucleotides characterized by 2'5'-phosphodiester bonds, designated 2'5'-oligo(A) or 2-5A (ref. 1). These oligonucleotides activate an endoribonuclease that degrades RNA in extracts of control and interferon-treated cells. These observations have been made in tissue culture cells and no informatin is yet available on these enzymatic activities in animals with elevated interferon levels. We report here on 2-5A synthesis in tissue homogenates and serum of mice infected with encephalomyocarditis virus (EMCV); this virus induces interferon synthesis when injected intraperitoneally into mice. Significant synthesis of 2-5A was detected in extracts of spleen and lungs, but also, surprisingly, in the serum of these mice. Subsequent experiments showed synthesis of 2-5A in serum of mice treated with the interferon inducer poly(I) x poly(C) (ref. 3) or with mouse fibroblast interferon.