The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

Principles of protein folding--a perspective from simple exact models.

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.     

水稻OsPR10A的表达特征及其在干旱胁迫应答过程中的功能

利用免疫印迹(WB)分析了水稻(Oryza sativa) OsPR10A在其不同生长时期、不同组织部位及多种非生物逆境胁迫下的表达特征, 发现OsPR10A在干旱、盐胁迫以及茉莉酸甲酯(MeJA)和脱落酸(ABA)诱导下表达量明显升高, 表明该蛋白可能在干旱和盐胁迫应答过程中发挥作用。为证明这一推测, 我们构建了OsPR10A超表达载体, 经农杆菌介导转化水稻, 获得超表达OsPR10A的纯合株系。田间表型观察表明, 转基因株系株高变矮、穗长变短、结实率降低。用20% PEG6000在水稻种子萌发过程中进行干旱处理, 结果显示, OsPR10A超表达株系的根长和芽长均显著高于野生型, 证明超表达OsPR10A可增强水稻萌发期耐旱性。该研究有助于增进人们对水稻OsPR10A功能的了解。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

氦氖激光对绵羊精清中酶活性效应的研究

本文研究结果表明低剂量的氦氖激光可以提高绵羊精清中GOT和LDH酶的活性,并对其机制作了初步的探讨。     

Quantitation of Rat Dopamine Transporter mRNA: Effects of Cocaine Treatment and Withdrawal

Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.     

The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.     

时域—频域结合分析法—一种分析果蝇求爱歌的新方法

我们设计了一种时域-频域结合分析法,并用此方法分析了6个种群12种果蝇的求爱歌,发现如果将时域与频域的研究结合起来,对求爱歌进行频谱分析,可以定量地揭示出求爱歌的频域特性及其在时域上的细微变化。我们还对果蝇求爱歌的时域模式进行了初步的探讨,发现它们是在同一基本成分上进行调制而产生的,亲缘关系较近的种具有相近的调制方式。在对杂交后代的求爱歌的频谱分析中,我们还发现频谱上的某些特点是能够遗传的。这一新的研究方法为果蝇的进化遗传学和神经遗传的研究提供了一种新的手段。     

氨基酸酯水解酶产生菌的筛选及其合成头孢立新的研究

From ten genera and 146 bacterial strains, 22 strains producing alpha-amino acid ester hydrolase were selected. Among them, AS 1.586 and 41-2 were the best. The optimal conditions for synthesis of cephalexin by pseudomonas aeruginosa 1.204 were investigated. The optimal pH and temperature for enzymatic synthesis reaction was pH 6.8 and 25 degrees C, respectively. By using 1% 7-ADCA, 3% PGME and 4% biomass, about 70% of 7-ADCA was converted to cephalexin under the mentioned conditions.