Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Medial septum glutamatergic neurons control wakefulness through a septo-hypothalamic circuit

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奈妥吡坦/β-环糊精包合物的制备、评价与表征

目的:制备奈妥吡坦/β-环糊精包合物,用以提高奈妥吡坦的水溶性.方法:采用饱和水溶液法,制备奈妥吡坦/β-环糊精包合物;以载药量为指标,考察奈妥吡坦与β-环糊精的质量比(芯壁比)、包合温度、包合时间、搅拌速度的影响.基于单因素试验结果,采用正交设计实验对制备处方和工艺进行优化,得到最优奈妥吡坦/β-环糊精包合物,并对其...     

MIP-T3, a novel protein linking tumor necrosis factor receptor-associated factor 3 to the microtubule network

In this study, we report the identification of a novel tumor necrosis factor receptor-associated factor 3 (TRAF3)-interacting protein designated MIP-T3. MIP-T3 is a 83-kDa protein with no significant homology to known mammalian proteins. MIP-T3 mRNA and TRAF3 mRNA are ubiquitously expressed, and TRAF3 is the only TRAF protein to interact with MIP-T3. The MIP-T3-TRAF3 interaction requires the coiled-coil TRAF-N domain of TRAF3. To our knowledge, this is the first case of a TRAF-binding protein that interacts with a single member of the TRAF family specifically through a TRAF-N coiled-coil domain. MIP-T3 binds to Taxol-stabilized microtubules and to tubulin in vitro, and MIP-T3 recruits TRAF3 to microtubules when both proteins are overexpressed in HeLa cells. In a 293 cell line stably expressing CD40, TRAF3 is released from the TRAF3.MIP-T3 complex and recruited to the CD40 receptor upon CD40 ligand stimulation. MIP-T3 may provide a novel mechanism in sequestering TRAF3 to the cytoskeletal network.     

益气活血方对肝星形细胞（HSC）Ⅰ型胶原的表达影响的研究

研究益气活血方剂抑制HSC合成Ⅰ型胶原的作用和生物学机制。采用免疫性肝纤维化大鼠模型,制备不同造模时间的病理血清、正常血清和益气活血方药物血清培养肝星形细胞HSC（Hepatic stellate cells）激光共 焦显微镜定量分析Ⅰ型胶原的表达。结果显示：（1）正常大鼠血清培养继代HSC表达较多的Ⅰ型胶原。纤维化模型大鼠血清诱导HSC表达的Ⅰ型胶原低于正常大鼠,益气活血药物血清可抑制纤维化模型血清诱导的HSCⅠ型胶原的表达,P＜0.05。（2）3周模型血清培养的HSC中分别加入10^-6、10^-7和10^-8ngγ-干扰素表明;10^-6、10^-7ngγ-干扰素和益气活血方具有相同的抑制HSC表达Ⅰ型胶原的作用,10^-7ngγ－干扰素抑制作用次之,P＜0.01。（3）1％胎牛血清可使原代HSC表达低水平Ⅰ型胶原。结果表明:益气活血方剂能够抑制HSC合成Ⅰ型胶原,其作用机制是改变了HSC的生活状态。     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

CNTF对烧伤大鼠血清引起大鼠海马神经元细胞毒性的影响

应用整体和离体神经元培养,观察CNTF对烧伤大鼠海马神经元及烧伤血清引起海马神经元损伤的影响,结果表明,大鼠烧伤后海马组织神经元数目减少,NO含量升高;烧伤大鼠血清可引起培养的海马神经元细胞存活率下降,培养液中NO含量升高;CNTF能降低烧伤大鼠海马组织中NO的含量,保护海马神经元,并能提高培养的海马神经元的存活率,减少培养液中NO含量,其作用呈剂量依赖性;CNTF对神经元存活率的影响与NO含量呈显著负相关,提示CNTF对烧伤大鼠血清引起的海马神经元损伤有保护作用,其作用机制可能是通过抑制NO的神经毒性。     

Definition of endotoxin binding sites in horseshoe crab factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides.

Three truncated fragments, harboring different sushi domains, namely, sushi123, sushi1, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins. Sushi1 and 3 each has a high-affinity LPS binding site with K:(d) of 10(-9) to 10(-10) M. Positive cooperativity in sushi123 resulted in a 1000-fold increase in K:(d)2. The core LPS binding region of sushi1 and 3 reside in two 34-mer peptides, S1 and S3. A rigidly held disulfide-bonded structure is not essential but is important for LPS binding, as confirmed by a 100- to 10000-fold decrease in affinity. Both S1 and S3 can inhibit LAL reaction and LPS-induced hTNF-alpha secretion with different potency. LAL assay revealed that at least two molecules of S1 bind cooperatively to one LPS molecule, with Hill's coefficient of 2.42. The LPS binding by S3 is independent and noncooperative. The modified SDelta1 and SDelta3 peptides exhibited increased LPS neutralization potential although its LPS binding affinities indicated only a 10-fold improvement. Hence, the structural difference of the four sushi peptides conferred different efficiencies in LPS neutralization without altering their binding affinity for LPS. Circular dichroism spectrometry revealed that the four peptides underwent conformational change in the presence of lipid A, transitioning from a random coil to either an alpha-helical or beta-sheet structure. Two factors are critical for the sensitivity of Factor C to LPS: 1) the presence of multiple binding sites for LPS on a single Factor C molecule; and 2) high positive cooperativity in LPS binding. The results showed that in the design of an improved LPS binding and neutralizing peptide, charge balance of the peptide is a critical parameter in addition to its structure.