Role of conserved transmembrane cationic amino acids in the prostaglandin transporter PGT

The prostaglandin transporter "PGT" interacts electrostatically with its anionic substrate, based on inhibition by the disulfonic stilbenes [Chan, B. S. (1998) J. Biol. Chem. 273, 6689-6697], inhibition by the thiol-reactive anion sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570], and the requirement for a negatively charged 1-position carboxyl on the substrate [Itoh, S. (1996) Mol. Pharm. 50, 736-742]. Here we found that modification of positively charged residues on wild-type PGT by arginine- and lysine-specific reagents significantly inhibited transport. We previously found that the binding site of PGT is formed, at least in part, by its membrane-spanning segments [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570]. Three charged residues within predicted transmembrane spans (E78, R560, and K613) are conserved in PGT and in related transporters. Substitution of the anionic residue E78 (E78D and E78C) produced an essentially functional transporter, whereas substitution of the cationic residues with neutral residues (R560N and K613Q) resulted in poorly functional transporters. Immunoblotting revealed similar expression levels of wild-type and mutant transporters, and immunostaining indicated correct targeting. Conservative charge substitutions (R560K, K613R, and K613H) resulted in generally functional transporters. In contrast, R560N was nonfunctional, whereas the substrate affinity of K613G decreased greater than 50-fold. Conservative substitutions retaining the charge at position 613 (K613R and K613H) restored the substrate affinity, suggesting a direct role of K613 in substrate binding. Double-neutral mutants E78G/R560C and E78G/K613C were inactive, indicating that these residues are not simply charge-paired. Our results suggest that an arginine at position 560 is critical for maximal substrate translocation, and that a positively charged side chain at position 613 contributes to electrostatic binding of the anionic substrate.     

我国登革3型病毒广西80-2株基因组全序列分析

对我国登革 3型病毒 80 2株基因组进行全序列测定 ,为了解其基因组结构与功能的关系提供依据 .根据登革 3型病毒H87株的序列设计并合成引物 ,应用RT PCR和RACE法 ,对 80 2株基因组RNA进行扩增、克隆测序后获得我国登革 3型病毒广西株基因组序列 .该株病毒基因组全长10 696nt ,不含poly(A)尾 ,4种碱基数分别为A :3 4 3 7,C :2 2 15,G :2 773 ,U :2 2 71.包含一个读码框架 ,自 95至 10 2 67位 ,共 10 170个碱基 ,编码 3 3 90个氨基酸 ,5′和 3′非编码区长度分别为 94nt和4 3 2nt.与H 87株比较 ,核苷酸和氨基酸序列同源性均在 99%以上 ,有 2 8个碱基发生改变 ,其中 2 6个碱基突变发生在读码框架内 ,碱基转换 18个 ,颠换 10个 ;碱基突变引起 14个氨基酸的改变 .80 2株与H87株病毒的基因组全序列同源性高 ,变异度小 .     

Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.     

t-PA昆虫细胞表达及特性分析

应用RT-PCR技术,从人黑色素瘤Bowes细胞株中扩增出人组织型纤溶酶原激活剂(tissue-typeplasminogenactivator,t-PA)cDNA。序列分析证实,与国外的报道完全一致。将含完整阅读框架的人t-PAcDNA克隆至昆虫细胞表达转移载体pBacPAK8中,获得重组质粒pBac-tPA。应用脂质体共转染法,将pBactPA和线性化杆状病毒BacPAK6DNA共转染Tn-5B-1昆虫细胞。经空斑筛选获得11株重组病毒。经PCR鉴定与生物活性测定,获得一株高效表达t-PA的重组病毒BactPA3。在含胎牛血清的培养基中,t-PA表达活性在感染(MOI≈10)后72h左右达到最高,为3.04×103IU／ml,即相当于1.8×104IU／106细胞;在无血清培养基中,t-PA最高表达水平相差不大,但时间为感染后132h左右。经SDS-PAGE纤维蛋白自显影分析,分子量为68kda左右。与从人黑色素瘤细胞培养液中提纯的天然t-PA相比,其受纤维蛋白原激活的特性、和纤维蛋白的亲和力及在血浆中的失活速率基本相同。表达的t-PA在血液循环中的半衰期为7min。     

3种小麦细胞质雄性不育系及其杂种线粒体DNA的RFLP分析

对细胞质分别来源于提莫菲维（Ｔ．ｔｉｍｏｔｈｅｅｖｉｉ）,粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）,偏凸山羊草（Ａｅ．ｖｅｎｙｒｉｃｏｓａ）的３种普通小麦的雄性不育系,相应保持系和恢复系及其上的ｍｔＤＮＡ用１２个线粒体基因探针进行了ＲＦＬＰ分析,结果为：⑴Ｔ、Ｋ、Ｖ型不育系的ｍｔＤＮＡ在组织结构上存在显著差异;⑵Ｔ、Ｋ、Ｖ不育系的ｍｔＤＮＡ与共同的保持系间显著不同,失测ｍｔＤＮＡ与小麦ｃｍｓ有关;⑶在     

Self-splicing of the Chlamydomonas chloroplast psbA introns.

We used alpha-32P-GTP labeling of total RNA preparations to identify self-splicing group I introns in Chlamydomonas. Several RNAs become labeled with alpha-32P-GTP, a subset of which is not seen with RNA from a mutant that lacks both copies of the psbA gene. Hybridization of the GTP-labeled RNAs to chloroplast DNA indicates that they originate from the psbA and rrn 23S genes, respectively, the only genes known to contain group I introns in this organism. Introns 1, 2, and 3 of psbA (with flanking exon sequences) were subcloned and transcribed in vitro. The synthetic RNAs were found to self-splice; splicing required Mg2+, GTP, and elevated temperature. In addition, the accuracy of self-splicing was confirmed for introns 1 and 2, and intermediates in the splicing reactions were detected. These results, together with our recent data on the 23S intron, indicate that the ability to self-splice is a general feature of Chlamydomonas group I introns. These findings have significant implications for the mechanism of group I intron splicing and evolution in Chlamydomonas and other chloroplast genomes.     

电场作用条件与作物种子生长的相关性

在综合评价电场强度及处理时间对作物生物效应影响时, 如何确定生物效应指标之间的权重一直是困扰生物信息综合评价的核心问题。比如叶绿素增长1 mg·L^–1 的价值相当于多少厘米根长的增加, 这是一个难以回答的问题。同时, 通过指标加权合成的方法获得综合评价的结果也存在信息丢失的缺陷。针对电场条件对作物生物效应的影响, 利用数据包络分析的基本思想, 给出了用于综合评价电场强度及处理时间对作物生物效应影响的非参数综合分析方法和相应的数学模型, 该方法不仅可以克服上述缺点, 而且还为分析不同外部条件对作物综合生物效应的影响程度提供了一种有效的分析工具。应用该方法综合评价了不同电场强度和不同处理时间对小麦 (Triticum aestivum)种子幼苗株高和根长的影响。结果表明: 不同电场强度和处理时间对小麦幼苗生长产生不同影响, 在0.5–6.0 kV·cm^–1场强范围内, 随电场强度增加, 生理指标呈现振荡性变化, 当处理时间为5分钟时, 1.0和2.0 kV·cm^–1场强为最佳处理条件; 处理时间为10分钟时, 2.5 kV·cm^–1场强为最佳处理条件。     

植物开花的组蛋白甲基化调控分子机理

开花是指植物从营养生长转变到生殖生长的生理过程, 是植物个体发育和后代繁衍的中心环节, 既受遗传基础决定,同时又受到温度和光周期等多种环境因素的调控。在拟南芥中, 已经分离了大量的与开花相关的基因, 从遗传学上已初步形成了一个开花调控的网络。组蛋白甲基化是植物发育过程的重要调节方式, 近年来关于其参与开花调控的研究有了重要进展。本文综述了具有代表性的组蛋白H3赖氨酸甲基化修饰参与调控植物开花发育的机制, 提出该研究领域的发展方向和前景。     

Virus variation resources at the National Center for Biotechnology Information: dengue virus

Background  

There is an increasing number of complete and incomplete virus genome sequences available in public databases. This large body of sequence data harbors information about epidemiology, phylogeny, and virulence. Several specialized databases, such as the NCBI Influenza Virus Resource or the Los Alamos HIV database, offer sophisticated query interfaces along with integrated exploratory data analysis tools for individual virus species to facilitate extracting this information. Thus far, there has not been a comprehensive database for dengue virus, a significant public health threat.     

Hypoxia suppresses KV1.5 channel expression through endogenous 15-HETE in rat pulmonary artery

Hypoxia initiated pulmonary vasoconstriction is due to the inhibition of voltage-gated K(+) (K(V)) channels. But the mechanism is unclear. We have evidence that hypoxia activates 15-lipoxygenase (15-LOX) in distal pulmonary arteries and increases the formation of 15-hydroxyeicosatetraenoate (15-HETE). 15-HETE-induced pulmonary artery constriction to be through the inhibition of K(V) channels (K(V)1.5, K(V)2.1 and K(V)3.4). However, no direct link among hypoxia, 15-HETE and inhibition of K(V) subtypes is established. Therefore, we investigated whether 15-LOX/15-HETE pathway contributes to the hypoxia-induced down-regulation of K(V) channels. As K(V)1.5 channel is O(2)-sensitive, it was chosen in the initial study. We found that inhibition of 15-LOX suppressed the response of hypoxic pulmonary artery rings to phenylephrine. The expressions of K(V)1.5 channel mRNA and protein was robustly up-regulated in cultured PASMC and pulmonary artery after blocking of 15-LOX by lipoxygenase inhibitors in hypoxia. The 15-LOX blockade also partly rescued the voltage-gated K(+) current (I(K(V))). 15-HETE contributes to the down-regulation of K(V)1.5 channel, inhibition of I(K(V)) and increase of native pulmonary artery tension after hypoxia. Hypoxia inhibits K(V)1.5 channel through 15-LOX/15-HETE pathway.