Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.     

Domain formation by a Rhodococcus sp. biosurfactant trehalose lipid incorporated into phosphatidylcholine membranes

The study of the interaction of biosurfactants with biological membranes is of great interest in order to gain insight into the molecular mechanisms of their biological actions. In this work we report on the interaction of a bacterial trehalose lipid produced by Rhodococcus sp. with phosphatidylcholine membranes. Differential scanning calorimetry measurements show a good miscibility of the glycolipid in the gel state and immiscibility in the fluid state, suggesting domain formation. These domains have been visualized and characterized, for the first time, by scanning force microscopy. Incorporation of trehalose lipid into phosphatidylcholine membranes produces a small shift of the antisymmetric stretching band toward higher wavenumbers, as shown by FTIR, which indicates a weak increase in fluidity. The CO stretching band shows that incorporation of trehalose lipid increases the proportion of the dehydrated component in mixtures with the three phospholipids at temperatures below and above the gel to liquid-crystalline phase transition. This dehydration effect is also supported by data on the phospholipid PO stretching bands. Small-angle X-ray diffraction measurements show that in the samples containing trehalose lipid the interlamellar repeat distance is larger than in those of pure phospholipids. These results are discussed within the frame of trehalose lipid domain formation, trehalose lipid/phospholipid interactions and its relevance to membrane-related biological actions.     

Roles of basic residues and salt-bridge interaction in a vacuolar H-pumping pyrophosphatase (AVP1) from Arabidopsis thaliana

To investigate the possible role of basic residues in H⁺ translocation through vacuolar-type H⁺-pumping pyrophosphatases (V-PPases), conserved arginine and lysine residues predicted to reside within or close to transmembrane domains of an Arabidopsis thaliana V-PPase (AVP1) were subjected to site-directed mutagenesis. One of these mutants (K461A) exhibited a “decoupled” phenotype in which proton-pumping but not hydrolysis was inhibited. Similar results were reported previously for an E427Q mutant, resulting in the proposal that E427 might be involved in proton translocation. However, the double mutant E427K/K461E has a wild type phenotype, suggesting that E427 and K461 form a stabilising salt bridge, but that neither residue plays a critical role in proton translocation.     

Association of putative ammonium exporters Ato with detergent-resistant compartments of plasma membrane during yeast colony development: pH affects Ato1p localisation in patches

It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.     

Biophysical analysis of the EPEC translocated intimin receptor-binding domain

Enteropathogenic Escherichia coli (EPEC) are Gram (-) bacteria responsible for widespread illness in the form of diarrhea. EPEC cells attach to the intestinal epithelium using a Type III secretion system common to many Gram (-) bacteria. The translocated intimin receptor (TIR) is the first protein secreted through the EPEC secretion complex, and is absolutely required for pathogenesis. It inserts into the intestinal epithelium, serving as an anchor responsible for the attachment of EPEC to the host epithelial cell. Intimin is a transmembrane protein displayed on the EPEC cell surface with an extracellular domain that binds TIR. Observation of a TIR-TIR dimer in the X-ray co-crystal structure of the extracellular domains of intimin and TIR raised the question of how these protein domains interact and function in solution. Herein we report that the extracellular domain of TIR exists in a folded and active monomeric state in solution, as confirmed by analytical ultracentrifugation, analytical size-exclusion HPLC, isothermal titration calorimetry, and surface plasmon resonance.     

Temporal consistency in the long-term spatial distribution of macroinvertebrate drift along a stream reach

Previous studies of the spatial pattern of stream invertebrate drift have focused on spatial variation at microhabitat scales or landscape scales, or temporal variation over diel or seasonal scales. None have examined consistency in spatial variation over longer time scales (>1 year). This study examined invertebrate drift density and composition at fixed locations (terminal ends of 10 riffles) each month at day and night along a 1 km reach of a 2nd order stream over a period of nearly 2 years. Consistent differences in the density of macroinvertebrate drift between riffles over 2 years were observed. The only habitat characteristic observed to be related to invertebrate drift density was the length and size of riffles above sampling sites, with larger and longer riffles producing the highest drift densities. Consistent differences in the supply of drifting macroinvertebrates along a stream reach may have implications for the supply of colonists to substrate patches and the profitability of feeding positions for drift-feeding fish and other predators. Handling editor: D. Dudgeon     

施硫对‘豫麦50’籽粒灌浆特性及产量的影响

选用弱筋小麦品种‘豫麦50’为材料,通过大田试验研究了较高供氮水平下(施纯氮330 kg/hm2)施用低量(S 20 kg/hm2)、中量(S 60 kg/hm2)和高量(S 100 kg/hm2)硫肥对小麦籽粒灌浆特性和产量的影响.结果显示,不同施硫处理的小麦籽粒灌浆进程均“呈慢—快—慢”的S型曲线,拟合的Logistic方程决定系数均大于0.993 2,并达到极显著水平;灌浆持续期(T)、渐增期平均灌浆速率(V1),快增期和缓增期持续期(T2、T3)、灌浆期各阶段干物质积累量(m1、m2、m3)和理论最大千粒重(K)均随施硫量增加而提高,并以中硫处理的各项灌浆参数表现较优;中硫处理的千粒重(46.47 g)和籽粒产量(7 416.0 kg/hm2)显著高于低硫处理,极显著高于对照(不施硫)和高硫处理.表明在当前较高施氮水平下,每公顷施纯硫60 kg可有效改善小麦灌浆特性,显著提高籽粒产量.     

毛柄小勾儿茶伴生群落种类组成及多样性研究

通过野外实地调查对小勾儿茶变种毛柄小勾儿茶(Berchemiella wilsoniivar. pubipetiolata)伴生群落4个分布点(浙江省临安市湍口和马啸、安徽省霍山县马家河和舒城县万佛山)的种类组成及物种多样性进行研究。结果表明:(1)种类丰富,但含多属或多种的大科、大属的优势地位不明显,极小科(仅含1属)、极小属(仅含1种)所占比例大;(2)属的北温带分布类型最多,科的世界分布类型比例大,仅次于泛热带分布类型;(3)4个分布群落乔木层、灌木层、草本层优势度均很低;灌木层Shannon-Wiener多样性指数较大,在4.13~4.49之间,乔木层和草本层较低。湍口、马家河、万佛山以灌木层Heip均匀度指数最大,马啸以乔木层均匀度指数最大;(4)马家河与万佛山之间的植物种类组成最相近,湍口与马啸也较相近;乔木层种类组成差异最大,灌木层最小。     

杨树粒肩天牛的生物学特性

杨树粒肩天牛在福建省2a发生1代;老熟幼虫在第2年4月下旬至5月初开始化蛹,成虫出现期始于5月底6月初,6月中下旬到7月上旬为盛发期,9月上旬只见个别成虫;产卵期始于6月中旬,6月下旬至7月中旬为产卵高峰期;幼虫孵化期为6月下旬到8月中旬,7月上中旬为为盛发期;幼虫不越冬。杨树粒肩天牛成虫为补充营养所取食的树种较集中,主要是构树与桑树。杨树粒肩天牛幼虫对杨树危害严重,但成虫却不喜欢取食杨树,用杨树饲养的粒肩天牛成虫寿命很短,仅3-19d,不产卵;而以桑树为补充营养的雌成虫的平均寿命为55d、雄26.5d;以构树为补充营养的雌成虫的平均寿命为78d、雄45.5d,补充桑树与构树的粒肩天牛成虫均可正常产卵。雌雄成虫一生可多次交尾。雌虫有多次产卵现象,单雌每天产卵数量1-12粒不等。