The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.

The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.     

1H NMR identification of a beta-sheet structure and description of folding topology in putidaredoxin

Putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, is the physiological reductant of P-450cam, which in turn catalyzes the monohydroxylation of camphor by molecular oxygen. No crystal structure has been obtained for Pdx or for any closely homologous protein. The application of two-dimensional 1H NMR methods to the problem of structure determination in Pdx is reported. A beta-sheet consisting of five short strands and one beta-turn has been identified from distinctive nuclear Overhauser effect patterns. All of the backbone resonances and a majority of the side-chain resonances corresponding to protons in the beta-sheet have been assigned sequence specifically. The sheet contains one parallel and three antiparallel strand orientations. Hydrophobic side chains in the beta-sheet face primarily toward the protein interior, except for a group of three valine side chains that are apparently solvent exposed. The potential significance of this "hydrophobic patch" in terms of biological activity is discussed. The folding topology, as determined by the constraints of the beta-sheet, is compared with that of other [2Fe-2S] proteins for which folding topologies are known.     

Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.

The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.     

Effect of cellulases on spontaneous fusion of maize protoplasts

Summary The effect of protoplast-isolating enzymes on spontaneous fusion of maize protoplasts (Zea mays L. cv. Black Mexican Sweet) was investigated using a convenient ethidium bromide nuclear staining procedure. After 2–2.5 hour digestion in an enzyme solution containing 1% Cellulysin, 0.5% Rhozyme, and 0.02% Pectolyase Y-23, 50–75% of the protoplasts contained multiple nuclei. The cellulase Cellulysin was identified as the factor causing the spontaneous protoplast fusion; when Cellulysin was replaced by CELF cellulase, most protoplasts were uninucleate. Calcium and other components in the enzyme solution did not affect spontaneous fusion. Cellulysin also increased the percentage of multinucleate protoplasts from rice and asparagus suspensions. Presence of multiple nuclei might affect genetic manipulations involving protoplasts.     

基于形态学空间格局分析和最小累积阻力模型的城市生态安全格局构建——以厦门市为例

城市化进程的快速发展加剧了生态系统的退化。如何扭转生态系统的退化,同时满足人类日益增长的生态系统服务需求,成为当前的一个研究热点。生态安全格局的构建在一定程度上可平衡城市发展与生态环境保护之间的关系,对于保障区域生态安全、提升生态系统功能具有重大意义。以厦门市为例,基于“生态源地识别—阻力面构建—生态廊道提取”的基本框架构建陆域生态安全格局。结合生态系统服务重要性评价和形态学空间格局分析识别生态源地,该方法兼顾了生态结构和功能,使得所识别的生态源地更具全面性。选取土地利用类型、高程和坡度构建生态综合阻力面,并用人类居住合成指数修正生态综合阻力面,以减少主观赋值的影响,识别各土地利用类型内部的差异,使生态阻力面的构建更加合理。在此基础上通过最小累积阻力模型提取生态廊道,利用重力模型量化潜在生态廊道的相对重要性,并根据重力模型结果划分重要性等级。研究结果表明,厦门市的生态安全格局由14个生态源地、21条生态廊道、15个生态节点及若干个踏脚石所组成。生态源地主要集中在研究区的西部和北部,以林地和草地为主,面积合计为558.64 km²。生态廊道长约159.40 km,其...     

The novel type II toxin–antitoxin PacTA modulates Pseudomonas aeruginosa iron homeostasis by obstructing the DNA-binding activity of Fur

Type II toxin–antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes and are involved in diverse critical cellular functions such as defense against phages, biofilm formation, persistence, and virulence. GCN5-related N-acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. We here describe a group of GNAT-Xre TA modules widely distributed among Pseudomonas species. We investigated PacTA (one of its members encoded by PA3270/PA3269) from Pseudomonas aeruginosa and demonstrated that the PacT toxin positively regulates iron acquisition in P. aeruginosa. Notably, other than arresting translation through acetylating aminoacyl-tRNAs, PacT can directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit the expression of downstream iron-acquisition-related genes. We further showed that the expression of the pacTA locus is upregulated in response to iron starvation and the absence of PacT causes biofilm formation defect, thereby attenuating pathogenesis. Overall, these findings reveal a novel regulatory mechanism of GNAT toxin that controls iron-uptake-related genes and contributes to bacterial virulence.     

双特异抗体的研究进展

双特异抗体是指可以同时结合两个不同抗原或一个抗原不同表位的特殊抗体,目前已有3个双特异抗体批准上市,还有很多个双特异抗体处于临床或临床前研究阶段。文中就双特异抗体的发现、制备方法、结构类型和设计策略、作用机制以及目前研究现状进行综述。     

Resveratrol induces AMPK and mTOR signaling inhibition-mediated autophagy and apoptosis in multiple myeloma cells

Resveratrol,a natural compound extracted from the skins of grapes,berries,or other fruits,has been shown to have anti-tumor effects against multiple myeloma(MM)...