An Electrostatically Preferred Lateral Orientation of SNARE Complex Suggests Novel Mechanisms for Driving Membrane Fusion

Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP(2)) is the most abundant phosphoinositide in the plasma membrane of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP(2) for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP(2). We first showed that PIP(2) regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP(2) levels. Moreover, by controlling the number of PIP(2)-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP(2), where each of the four open states corresponds to the channel activated by one to four PIP(2) molecules.     

Synthesis of a coumarin based fluorescent amino acid

L-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid (L-Adp), as a non-proteinogenic fluorescent amino acid has been synthesized by a highly stereoselective routine (>99.5%). This fluorescent amino acid, as fluorophore-quencher pair, may be used to study peptide assays. For enantiomeric excess determination, the racemic D-Adp (D-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid) has also been synthesized.     

Cholesterol is an important factor affecting the membrane insertion of beta-amyloid peptide (A beta 1-40), which may potentially inhibit the fibril formation.

beta-Amyloid peptide (A beta), a normal constituent of neuronal and non-neuronal cells, has been proven to be the major component of extracellular plaque of Alzheimer's disease. Interactions between A beta and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. Here we show that A beta is able to insert into lipid bilayer. The membrane insertion ability of A beta is critically controlled by the ratio of cholesterol to phospholipids. In a low concentration of cholesterol A beta prefers to stay in membrane surface region mainly in a beta-sheet structure. In contrast, as the ratio of cholesterol to phospholipids rises above 30 mol%, A beta can insert spontaneously into lipid bilayer by its C terminus. During membrane insertion A beta generates about 60% alpha-helix and removes almost all beta-sheet structure. Fibril formation experiments show that such membrane insertion can reduce fibril formation. Our findings reveal a possible pathway by which A beta prevents itself from aggregation and fibril formation by membrane insertion.     

鄂西北武当山区野生植物资源调查及开发对策

新阶段推进农业结构战略性调整 ,增加农民收入 ,山区的亮点在于发挥比较优势 ,开发特色资源。本文运用市场经济价值法则 ,对武当山区的野生资源进行了初步调查和分类 ,并提出了科学开发的对策     

根系发育的营养调控及对其生境的影响

根系发育对土壤中无机营养的供应和分配的变化非常敏感,本文综述了N、P、K等营养影响根系的发育进程如分枝、根毛产生、直径、生长角度、结瘤作用和簇生根的形成等的机制。营养的供应对根系发育的影响既可以是直接的、是外部营养浓度改变的结果、也可以是间接的、是植物内部营养状况变化的结果。直接途径引起暴露在营养供应中的那部分根系的发育反应;间接途径引起系统的反应,并且似乎依赖于来自冠部的长距离的信使。讨论了最新所了解的内外营养感受(sensing)的机制,长距离信号的可能特征,激素在营养形态发生反应中的作用,根系性状的基因型差异和遗传特性,以及植物根系在生态恢复、防止环境污染、全球气候变化和资源可持续发展中的作用。     

环境因子对苋菜碳、氮代谢关键酶及抗氧化酶活性的影响

研究了塑料大棚内光照强度、气温、地温、二氧化碳浓度及氮肥施量等5个环境因子、5个水平处理组合对春苋菜早熟栽培生理的影响。结果表明,在碳、氮代谢关键酶方面,不同氮水平下,光强、气温与硝酸还原酶(NR)活性(极)显著正相关,各处理组合中其活性次序为A(0网0膜)>B(0网1膜)>C(1网1膜)>D(2网1膜)>E(3网1膜),后4者分别比处理A(对照)降低了6.45%、34.21%、61.54%和63.78%;CO2浓度、气温和地温(极)显著影响磷酸烯醇式丙酮酸羧化酶(PEPC)活性,处理B的PEPC平均活性最强,处理B、C、D、E的PEPC活性较A分别增加了59.69%、28.57%、-10.71%和-17.86%。在活性氧代谢方面,超氧化物歧化酶(SOD)活性与光强正相关,与CO2浓度负相关,大小表现为A>B>C>D>E;过氧化物酶(POD)活性与光强、气温(极)显著负相关,为AA>C>D>E。POD在处理A、B、C中起保护作用,而在D、E中可能起伤害作用。各环境因子组合中,B(0网1膜:平均光强17800lx、地温21.2℃、气温23.5℃及CO2浓度1500μmol.mol-1)在施氮水平为N5(20kg.667m-2)时,各酶活协调性最好,可以实现苋菜的高产、高效和优质栽培。     

Misexpression of MIA disrupts lung morphogenesis and causes neonatal death

Microarray experiments designed to identify genes differentially expressed in the E11.5 lung and trachea showed that melanoma inhibitory activity (Mia1) was expressed only in the lung. Mia1 was abundantly expressed during early lung development, but was virtually absent by the end of gestation. Distal embryonic lung epithelium showed high levels of Mia1 expression, which was suppressed by treatment with either retinoic acid or the FGF signaling antagonist SU5402. Late-gestation fetuses in which lung epithelial hyperplasia was induced by misexpression of FGF7 or FGF10 showed continued expression of Mia1 in areas of aberrant morphogenesis. Mia1 expression was also significantly increased in urethane-induced lung adenomas. Treatment of E18.5 lung explants with exogenous MIA caused significant reductions in the expression of the lung differentiation markers Sftpa, Sftpb, Sftpc, and Abca3. Bitransgenic mice expressing MIA under the control of the SFTPC promoter after E16.5, the age when Mia1 is normally silenced, died from respiratory failure at birth with morphologically immature lungs associated with reduced levels of saturated phosphatidylcholine and mature SP-B. Microarray analysis showed significant reductions in the expression of Sftpa, Sftpb, Abca3, Aqp5, Lzp-s, Scd2, and Aytl2 in lungs misexpressing MIA. These results suggest that the silencing of Mia1 that occurs in late gestation may be required for maturation of the surfactant system.