Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Retinoic acid exerts sexually dimorphic effects on muscle energy metabolism and function

The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/− mice. Relative to wild-type (WT) controls, Rdh10+/− males fed a high-fat diet decrease reliance on fatty-acid oxidation and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/− females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/− male GM decrease 38% relative to WT. Rdh10+/− male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity, and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/− female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity, and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.     

Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.     

Kinetic characterization of the double mutant R148A/E182S of glycogen phosphorylase kinase catalytic subunit: the role of the activation loop

Many protein kinases are activated by phosphorylation in a highly conserved region of their catalytic subunit, termed activation loop. Phosphorylase kinase is constitutively active without the requirement for phosphorylation of residues in the activation loop. The residue which plays an analogous role to the phosphorylatable residues in other protein kinases is Glu182, which makes contacts to a highly conserved Arg148. In turn, Arg148 adjacent to the catalytic Asp149, enabling information to be transmitted from the activation loop to the catalytic machinery. The double mutant R148A/E182S has been kinetically characterized. The mutation resulted in an approximate 16- to 22-fold decrease in the k _cat/K _m value of the enzyme. The kinetic data, discussed in the light of the structural data from previously determined complexes of the enzyme, lead to the suggestion that the activation loop has a major role in substrate binding but also in correct orientation of the groups participating in catalysis.     

水稻矮杆多分蘖突变体CA648的光合生理特性分析

本研究分析了水稻矮杆多分蘖突变体丛矮648 (CA648)及对照中花11 (ZH11)在分蘖期叶片的叶绿素含量、光合速率,还原糖、蔗糖含量以及POD酶活力。结果表明,分蘖期ZH11剑叶中叶绿素含量高于CA648。水稻分蘖前期,CA648的净光合速率、气孔导度、蒸腾速率都低于ZH11,胞间CO2浓度高于ZH11;而在分蘖盛期(分蘖期后20 d)两种材料的净光合速率差异显著,ZH11净光合速率比CA648高17.5%,同时CA648的气孔导度、胞间CO2浓度和蒸腾速率都低于ZH11;而分蘖后期ZH11与CA648净光合速率几乎相同。分蘖盛期CA648叶片还原糖含量明显比对照高;而茎杆还原糖含量与对照无差异。CA648叶片蔗糖含量低于对照,约为ZH11的77.5%;而茎杆的蔗糖含量高于对照,比ZH11高30.7%。两种材料POD酶活力差异明显,其中在分蘖盛期,CA648叶、茎中的POD酶活力分别比ZH11高73.0%、16.8%。     

CRISPR Primer Designer:Design primers for knockout and chromosome imaging CRISPR-Cas system

The clustered regularly interspaced short palin-dromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regula...     

文心兰浅绿条纹突变体的生理生化及叶绿素荧光特性研究

以文心兰浅绿条纹突变体为材料,分析叶片光合色素含量和组成、叶绿素合成前体物质含量以及叶绿素荧光参数的变化,观察突变体叶绿体超微结构的改变,以探寻其叶色变异的生理基础。结果表明:(1)突变体叶绿素a(Chl a)、叶绿素b(Chl b)、类胡萝卜素(Car)和总叶绿素(Chl)含量分别比叶色正常植株显著降低了37.1%、34.0%、30.8%和36.3%。(2)突变体叶绿素生物合成受阻于胆色素原(PBG)到尿卟啉原Ⅲ(UrogenⅢ)的反应步骤。(3)突变体叶绿体发育存在明显的缺陷,基粒数目及基粒片层的垛叠层数明显减少,嗜锇颗粒及囊泡较多。(4)突变体初始荧光(Fo)比正常植株高39%,最大荧光(Fm)、最大光化学效率(Fv/Fm)、PSⅡ有效光化学效率(Fv′/Fm′)和PSⅡ实际光化学效率(ΦPSⅡ)均显著低于正常植株,但光化学淬灭系数(qP)和非光化学淬灭系数(NPQ)与正常植株无显著差异。研究结果说明,文心兰叶绿素生物合成受阻和叶绿体结构发育不良,导致叶绿素的含量下降,致使突变体叶片呈现浅绿条纹,光能利用率降低。     

无乳链球菌鱼源株10 kb基因序列对细菌致病力的影响

【目的】在前期比较基因组学分析中,我们发现中国无乳链球菌鱼源株GD201008-001基因组中有一段10 kb基因序列,内含11个未知功能的开放阅读框。为了研究该段基因序列与细菌的致病力的关系,本研究将这段基因进行了全段缺失。【方法】运用链球菌-大肠杆菌穿梭质粒p SET4s,构建了10 kb基因缺失株(Δ10 kb),并通过生物学性状的比较,细胞粘附试验,斑马鱼攻毒试验和缺失前后毒力相关基因转录水平的检测,评价该序列对无乳链球菌毒力的影响。【结果】经测序证明缺失株Δ10 kb构建成功,与亲本株GD201008-001相比较,缺失株Δ10 kb在细菌染色形态、对HEp-2细胞的粘附能力无明显差异,但在培养液中的生长速度略慢;缺失株Δ10 kb对斑马鱼的毒力明显增强,LD_(50)有极其显著的差异(P0.001);编码菌毛骨架蛋白2b的基因(PI-2b)和唾液酸酶基因(neul)在缺失株中的转录水平明显上升。【结论】该序列对无乳链球菌GD201008-001的毒力有显著的影响,可能调控某些毒力基因的转录表达,使细菌的毒力减弱。