KIF1A alternately uses two loops to bind microtubules

The motor protein kinesin moves along microtubules, driven by adenosine triphosphate (ATP) hydrolysis. However, it remains unclear how kinesin converts the chemical energy into mechanical movement. We report crystal structures of monomeric kinesin KIF1A with three transition-state analogs: adenylyl imidodiphosphate (AMP-PNP), adenosine diphosphate (ADP)-vanadate, and ADP-AlFx (aluminofluoride complexes). These structures, together with known structures of the ADP-bound state and the adenylyl-(beta,gamma-methylene) diphosphate (AMP-PCP)-bound state, show that kinesin uses two microtubule-binding loops in an alternating manner to change its interaction with microtubules during the ATP hydrolysis cycle; loop L11 is extended in the AMP-PNP structure, whereas loop L12 is extended in the ADP structure. ADP-vanadate displays an intermediate structure in which a conformational change in two switch regions causes both loops to be raised from the microtubule, thus actively detaching kinesin.     

Molecular architecture of the rotary motor in ATP synthase

Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion. Its membrane-embedded F0 sector has a rotation generator fueled by the proton-motive force, which provides the energy required for the synthesis of ATP by the F1 domain. An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c subunit forms an alpha-helical hairpin. The interhelical loops of six to seven of the c subunits are in close contact with the gamma and delta subunits of the central stalk. The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.     

拟南芥抗菌核病突变体275-7的初步研究

草酸是核盘菌致病过程中产生的毒素因子。以菌核病菌毒素草酸（3 mmol/L）为筛选压,从1 000个拟南芥T-DNA插入突变体中筛选出了1个草酸不敏感突变体,命名为275-7。通过拟南芥活体接种对突变体275-7进行菌核病抗性鉴定,与野生型相比,突变体275-7对菌核病的抗性显著增强（P<0.05）;荧光定量PCR检测结果表明,275-7中茉莉酸途径的标志基因PDF1.2的表达量是野生型的12倍,而水杨酸途径的标志基因PR1基因的表达量与野生型比较无差异。推测拟南芥突变体275-7可能通过增强水杨酸介导的防卫反应,从而表现出对菌核病的抗性。     

Atmospheric Lifetime of CHF2Br, a Proposed Substitute for Halons

The rate coefficients, k(1), for the reaction of OH with CHF(2)Br have been measured using pulsed photolysis and discharge flow techniques at temperatures (T) between 233 and 432 K to be k(1), = (7.4 +/- 1.6) x 10(-13) exp[-(1300 +/- 100)/T] cubic centimeters per molecule per second. The ultraviolet absorption cross sections, sigma, of this molecule between 190 and 280 nanometers were measured at 296 K. The k(1), and sigma values were used in a one-dimensional model to obtain an atmospheric lifetime of approximately 7 years for CHF(2)Br. This lifetime is shorter by approximately factors of 10 and 2 than those for CF(3)Br and CF(2)ClBr, respectively. The ozone depletion potentials of the three compounds will reflect these lifetimes.     

锰、铜、锌、铁添加水平对产蛋鸡生产性能和养分表观利用率的影响

设计锰、铜、锌、铁不同添加水平的A,B,C,D和E 5组日粮(A组为不添加4种微量元素的基础日粮;B组添加锰14 mg/kg;C组添加锰34 mg/kg;D组添加锰54 mg/kg、铜2 mg/kg、锌13 mg/kg;E组添加锰10mg/kg、铜2 mg/kg、锌17.5 mg/kg、铁22.5 mg/kg)分别饲喂5组275日龄罗曼褐壳蛋鸡,每组6个重复,每重复15只,试验期60 d,研究锰、铜、锌、铁不同添加水平对产蛋鸡生产性能和养分表观利用率的影响。结果表明,不添加4种微量元素的基础日粮(A组)的产蛋性能和养分利用率较高;锰是影响鸡产蛋率、产蛋量和其他元素吸收率的主要因素,添加锰使生产性能和养分利用率降低,锰添加量为34 mg/kg时产蛋率和能量表观利用率显著降低(P<0.01),日粮中锰和表观可利用锰的推荐含量应分别低于46和14 mg/kg;基础日粮中添加铜、锌和铁不影响蛋鸡生产性能和能量、粗蛋白质、钙和磷的表观利用率(P>0.05)。说明基础日粮中锰和铁已能满足或超过蛋鸡营养需要,添加锰对生产不利,而基础日粮中铜和锌相对缺乏。     

禽呼肠孤病毒σ3基因在烟草中的表达与鉴定

【目的】研究禽呼肠孤病毒S1基因编码的σ3基因在烟草中的表达及其表达产物的反应原性。【方法】根据Gen Bank中公布的禽呼肠孤毒株S1133的基因序列,设计特异引物扩增σ3基因,构建重组植物表达载体p BI121-σ3,热激法转化农杆菌EHA105感受态细胞,筛选含有重组质粒的p BI121-σ3的阳性工程菌株EHA105。采用农杆菌介导的叶盘法转化野生型烟草,卡那霉素筛选获得抗性植株。随后通过σ3基因的特异引物,PCR方法初步筛选获得转σ3基因的烟草植株。进一步对阳性植株通过实时荧光定量PCR方法检测σ3基因在转基因烟草中的拷贝数,以烟草自身单拷贝的内源基因核糖核酸还原酶-RNR2作为内参基因,通过梯度稀释分别构建RNR2及σ3基因的起始模板量和对应CT值的标准曲线,通过检测样品中σ3基因和RNR2基因模板量对数值的比值估算出σ3基因在转基因烟草中的拷贝数。并通过Western-blotting方法对转基因烟草中表达的σ3融合蛋白的反应原性进行分析。【结果】1通过双酶切和测序验证表明σ3基因插入位置、大小和读码框均正确,表明σ3基因已经成功的构建于植物表达载体p BI121的花椰菜花叶病毒(Ca MV)35S启动子的下游,以NOS为终止子,含有卡那霉素抗性标记(NPT II)。其表达的蛋白与下游的绿色荧光蛋白形成融合蛋白,其大小约为61.6 ku;2在转化筛选抗性植株的过程中采用350 mg·L-1的头孢霉素可以很好地抑制农杆菌的污染,100 mg·L-1的硫酸卡那霉素筛选压力能够很好地抑制非转化细胞的生长;3随机选取10株转化烟草进行PCR检测发现8株转化烟草可以扩增到清晰的目的条带约994 bp;4内参基因RNR2的标准曲线为y=-3.5352x+15.143,σ3基因的标准曲线为y=-3.5366x+1.8265,两个标准曲线的相关系数均接近于1。在检测的5株转化烟草中σ3基因的拷贝数均为4,阴性对照没有扩增。5Western-blotting显示σ3融合蛋白在转化烟草中可以正确表达,目的条带大小约为61.6 k D,与预计大小一致;σ3融合蛋白能够与禽呼肠孤病毒的阳性血清发生特异反应,表明该融合蛋白具有良好的反应原性。【结论】成功地构建了植物表达载体p BI121-σ3,筛选获得低拷贝的转σ3基因的烟草,σ3融合蛋白能够在烟草中表达且具有相应的反应原性,为进一步研究σ3基因的功能和利用植物作为生物反应器生产σ3蛋白口服疫苗的研发奠定了基础。     

不同来源ATP表征冷鲜羊肉新鲜度

【目的】通过系统研究冷鲜羊肉不同来源的三磷酸腺苷（adenosine triphosphate,ATP。包括肉中ATP、微生物ATP、肉表面ATP）在贮藏期间的变化规律,筛选能够表征冷鲜羊肉新鲜度变化的ATP指标,构建菌落总数和挥发性盐基氮预测模型,探究冷鲜羊肉新鲜度的预测新方法。【方法】以小尾寒羊背最长肌为试验材料,在空气密封包装0℃条件下分别贮藏0、1、3、5、7、9、11、13、15、17和21 d,分析冷鲜羊肉贮藏期间新鲜度指标（pH、色泽、挥发性盐基氮、菌落总数）与3种来源ATP（肉中ATP、微生物ATP、肉表面ATP）的变化,利用数据统计评价不同来源ATP的变化规律,并构建新鲜度指标的预测模型。【结果】冷鲜羊肉贮藏期间新鲜度指标菌落总数、挥发性盐基氮均呈现上升趋势,并均在贮藏17 d时超过国家标准限值;肉中ATP呈现不断下降趋势,微生物ATP与肉表面ATP均呈现上升趋势,与新鲜度指标变化趋势保持一致;冷鲜羊肉贮藏期间,肉中ATP、微生物ATP、肉表面ATP含量与菌落总数、挥发性盐基氮的相关系数（R）分别为-0.399、0.910、0.943和-0.357、0.725、0.9...     

超高压下Ca~(2+)与Na~+离子对甜菜果胶结构及流变性质的影响

[目的]研究超高压下不同浓度Ca2+与Na+对甜菜果胶结构及流变性质的影响,为甜菜果胶在食品中的应用提供理论依据。[方法]甜菜果胶用浓度0.05 mol·L-1的Tris-HCl溶液溶解,添加不同浓度Ca2+(2、12和20mmol·L-1)和Na+(0.05、0.1和0.6 mol·L-1),配制成1%(w/v)甜菜果胶溶液后进行超高压处理,然后分别对甜菜果胶分子量、微观结构、黏度和动态粘弹性进行测定。[结果]与常压下相比,在450 MPa条件下处理不同时间(10、20、30和50 min)后,甜菜果胶在1 550 cm-1处均出现新的吸收峰,甜菜果胶溶液的屈服应力σ0显著增加,但不同超高压处理时间之间无显著差异。添加不同浓度Ca2+或Na+的甜菜果胶在450 MPa条件下处理30 min,其结构及流变性的变化有所不同。相对于未添加Ca2+或Na+的甜菜果胶,添加2 mmol·L-1 Ca2+离子使甜菜果胶溶液屈服应力σ0、储能模量G’和损耗模量G"均明显增加,当Ca2+浓度增加到12 mmol·L-1和20 mmol·L-1时,果胶的流变性质变化不显著;添加2 mmol·L-1 Ca2+使甜菜果胶分子发生明显的交联。果胶分子量由只高压处理的2.25×105 Da显著增加到6.07×105 Da,Ca2+的添加浓度增加到20 mmol·L-1,果胶的分子量变为5.99×105 Da,与添加2mmol·L-1 Ca2+时没有显著差异,其流变性质变化亦不显著。相对于未添加Ca2+或Na+的甜菜果胶,添加0.05 mol·L-1 Na+也使甜菜果胶的屈服应力σ0显著增加,并且随着Na+浓度的持续增加,果胶的屈服应力σ0显著增加;而只有当Na+浓度增加到0.6 mol·L-1时,甜菜果胶储能模量G’和损耗模量G"才发生明显增加。添加0.1 mmol·L-1 Na+的甜菜果胶,其果胶分子链相互交联成网状,果胶分子发生明显聚集,果胶分子量显著增加到11.95×105 Da;而当Na+浓度增加到0.6 mol·L-1时,果胶链呈棒状结构,果胶分子量显著降低到5.53×105 Da。[结论]超高压下Ca2+与Na+可能与甜菜果胶分子结合使其结构发生改变,进而影响甜菜果胶的结构及流变性质。     

Atmospheric trends in methylchloroform and the global average for the hydroxyl radical

Frequent atmospheric measurements of the anthropogenic compound methylchloroform that were made between 1978 and 1985 indicate that this species is continuing to increase significantly around the world. Reaction with the major atmospheric oxidant, the hydroxyl radical (OH), is the principal sink for this species. The observed mean trends for methylchloroform are 4.8, 5.4, 6.4, and 6.9 percent per year at Aldrigole (Ireland) and Cape Meares (Oregon), Ragged Point (Barbados), Point Matatula (American Samoa), and Cape Grim (Tasmania), respectively, from July 1978 to June 1985. These measured trends, combined with knowledge of industrial emissions, were used in an optimal estimation inversion scheme to deduce a globally averaged methylchloroform atmospheric lifetime of 6.3 (+ 1.2, -0.9) years (1sigma uncertainty) and a globally averaged tropospheric hydroxyl radical concentration of (7.7 +/- 1.4) x 10(5) radicals per cubic centimeter (1sigma uncertainty). These 7 years of gas chromatographic measurements, which comprise about 60,000 individual calibrated real-time air analyses, provide the most accurate estimates yet of the trends and lifetime of methylchloroform and of the global average for tropospheric hydroxyl radical levels. Accurate determination of hydroxyl radical levels is crucial to understanding global atmospheric chemical cycles and trends in the levels of trace gases such as methane.     

Paramagnetic unit in spinach subchloroplast particles: estimation of size

A pulsed ruby laser (wavelength, 694.3 nanometers) was used to measure the dependence on light intensity of light-induced electron paramagnetic resonance (ERR) signal 1 for short flashes of uniform duration (400 microseconds). Approximately 10(18) photons per square centimeter per flash from the unattenuated beam were available to the sample of subchloroplast "system 1" particles from spinach. The experimental dependence of the EPR signal height plotted as a function of the total number of incident photons per flash was exponential. From measurement of the slope at a very low relative photon flux and the saturated EPR signal amplitude, the value for the cross section or "effective size" of the light-induced paramagnetic unit, sigma(EPR), was found to be 300 x 10(-17) square centimeter. This result is compared with a measured optical absorption cross section, sigma(694nm), of 2.5 x 10(-17) square centimeter, for the identical sample at the laser wavelength. The hundredfold difference in size supports the thesis that the paramagnetic state is a property of an aggregate of chlorophyll molecules of the same general size as the photosynthetic unit.     

原核表达番鸭呼肠孤病毒σC与σB蛋白对雏番鸭的免疫保护

【目的】探讨原核表达的番鸭呼肠孤病毒（DRV）外壳蛋白σC与σB对于雏番鸭的免疫保护作用,为研制有效的DRV亚单位疫苗提供理论基础。【方法】对pET-30a-S3/BL21重组菌与pET-30a-S4 ORF2/BL21重组菌进行稳定性检验,表达的重组蛋白进行纯化和复性后,进行油乳剂制备,50只1日龄雏番鸭随机平均分为5组,即DRV σB蛋白免疫组、DRV σC蛋白免疫组、DRV σB + σC蛋白免疫组及PBS对照组和攻毒对照组,免疫组皮下接种剂量为500 μg/只,7日龄时进行二次免疫,14日龄时进行攻毒试验,ELISA和琼扩（AGP）检测抗体水平并计算保护指数。【结果】重组菌具有良好的稳定性,各免疫组的雏番鸭均在第2次免疫后7 d产生了一定效价的抗体,其中DRV σB + σC蛋白免疫组和DRV σC蛋白免疫组的雏番鸭血清抗体ELISA效价均为1﹕200,高于DRV σB蛋白免疫组的1﹕100。攻毒保护试验结果表明,联合免疫的保护指数最高,可达70,重组DRV σC保护指数次之,为60,重组DRV σB蛋白保护指数最低,仅为40。【结论】使用原核表达的DRV外壳蛋白σC与σB制备油乳剂,联合免疫可诱导雏番鸭快速产生一定效价的抗体,并提供良好的免疫保护力。     

A role for interaction of the RNA polymerase flap domain with the sigma subunit in promoter recognition

In bacteria, promoter recognition depends on the RNA polymerase sigma subunit, which combines with the catalytically proficient RNA polymerase core to form the holoenzyme. The major class of bacterial promoters is defined by two conserved elements (the -10 and -35 elements, which are 10 and 35 nucleotides upstream of the initiation point, respectively) that are contacted by sigma in the holoenzyme. We show that recognition of promoters of this class depends on the "flexible flap" domain of the RNA polymerase beta subunit. The flap interacts with conserved region 4 of sigma and triggers a conformational change that moves region 4 into the correct position for interaction with the -35 element. Because the flexible flap is evolutionarily conserved, this domain may facilitate promoter recognition by specificity factors in eukaryotes as well.     

Crystal structure of a lipid G protein-coupled receptor

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P(1), resulting in the modulation of immune and stromal cell responses.     

ROSAT X-ray detection of a young brown dwarf in the chamaeleon I dark cloud

Photometry and spectroscopy of the object Cha Halpha 1, located in the Chamaeleon I star-forming cloud, show that it is a approximately 10(6)-year-old brown dwarf with spectral type M7.5 to M8 and 0.04 +/- 0.01 solar masses. Quiescent x-ray emission was detected in a 36-kilosecond observation with 31.4 +/- 7.7 x-ray photons, obtained with the Rontgen Satellite (ROSAT), with 9final sigma detection significance. This corresponds to an x-ray luminosity of 2.57 x 10(28) ergs per second and an x-ray to bolometric luminosity ratio of 10(-3.44). These are typical values for late M-type stars. Because the interior of brown dwarfs may be similar to that of convective late-type stars, which are well-known x-ray sources, x-ray emission from brown dwarfs may indicate magnetic activity.     

Isotopic composition of strontium in volcanic rocks from oahu

Analysis of several well-documented specimens from each of the three volcanic series on Oahu gives the following mean ratios of Sr(87) to Sr(86): the Waianae series, 0.7030 +/- 0.00010 (sigma); the Koolau series, 0.70385+/- 0.00009 (sigma); and the Honolulu series, 0.7029 ++/- 0.00006 ( sigma). The mean ratio of Sr(87) to Sr(86) of the Koolau series specimens is significantly higher than the means of the other two series. With one exception, significant differences in Sr(87)/ Sr(86) within a series were not found, even though some large compositional differences existed.     

原核生物RNA聚合酶辅助因子σ因子的研究进展

在原核生物中,σ因子是RNA聚合酶的辅助因子,被用来识别启动子中的功能序列,在基因的转录调控中发挥着重要的功能。目前原核模式生物中的可替换型σ因子大部分已被发现,其中大肠杆菌有4种,枯草芽胞杆菌有18种。从σ因子的分类、应答机制、识别元件、结构特征等方面进行了归纳。将为系统的理解原核生物σ因子的功能,及其在基因表达调控中的贡献等方面提供帮助。     

Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation

DNMT1, the major maintenance DNA methyltransferase in animals, helps to regulate gene expression, genome imprinting, and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methylcytosine is positioned within a shallow hydrophobic concave surface, whereas the cytosine on the target strand is looped out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated maintenance DNA methylation.