G蛋白在松类植物花粉管发育中的亚细胞定位及调控机制

G蛋白参与了哺乳动物内多种细胞信号途径，但其在植物花粉萌发和花粉管发育过程中的细胞学定位、生化特性及功能研究比较滞后，有关这方面的研究报道较少。在显花植物授粉受精过程中，具顶端极性生长特性的花粉管是雄性生殖单位的载体，也是研究细胞生长分子调控机理的理想体系。与被子植物相比，裸子植物具有生长周期长，花粉管生长缓慢、易分叉等特点，具有不同于被子植物花粉发育的独特发育模式。对于裸子植物花粉萌发和花粉管生长的调控机理，目前尚不十分清楚。本文以松类植物中比较有代表性的裸子植物青杆（Piceawillsonii）和白皮松(Pinus bungeana)花粉为试材，应用免疫分析和间接免疫荧光显微镜技术，结合药理学实验和FTIR手段，研究了异三聚体G蛋白和小G蛋白在花粉管细胞中的定位、生化特性及其在花粉管发育中的调控作用。结果如下： 应用Western Blotting技术和来自于抗哺乳动物中不同序列G蛋白O【亚基抗体，我们在白皮松花粉管中检测到一条分子量为40 kDa左右的蛋白。去污剂处理显示，该蛋白与质膜偶联。间接免疫荧光显微镜实验发现，在花粉管发育的整个时期，代表Ga蛋白的荧光均一的分布在整个质膜区域，尤其在尖端皮层区域荧光最亮，显示此处该蛋白浓度最高。无论是在正常发育的花粉管抑或是发生弯曲或扭曲生长的花粉管，均呈现同样的分布模式。随着花粉管发育，Ga蛋白表达量发生变化。在花粉管发育中期，Ga蛋白表达量比较高;随着花粉管离体培养时间的延长，Ga蛋白表达量下降。另外，在花粉刚刚萌发时，Ga蛋白表达量也比较低。 对白皮松花粉萌发进行的药理学实验显示，G蛋白调节剂 CTX和PTX对白皮松花粉管的影响呈现双阶段效应。当添加的药剂浓度小于400 ng mL-I时，无论CTX还是PTX均抑制了花粉萌发和花粉管生长，且花粉管容易破裂;而当二者浓度分别升至500 ng mL-I时，同对照相比，花粉管生长明显受到促进。这一结果不支持Ma等人在百合花粉中的研究结果。进一步应用FTIR技术分析发现，当用浓度为400 ng mL-I CTX或PTX处理花粉管时，花粉管细胞壁酚类物质增加，而纤维素、半纤维素、木聚糖等物质下降，这可能是导致此浓度处理下花粉管易破裂的原因。这些结果显示了G蛋白a亚基参与了白皮松花粉管生长，CTX和PTX可能通过下游对其敏感的功能蛋白而非Ga本身，影响着花粉管生长并调控着花粉管壁的建成。 利用来源于烟草的抗NtRacl抗体和拟南芥的抗ROPs抗体，应用WeternBlotting技术，我们在青杆花粉管中检测到分子量为23kDa的多肽。间接免疫荧光显微镜实验显示，在花粉萌发18和24小时后，Rac蛋白主要定位于花粉管尖端质膜区域，时而会延伸到顶端两侧区域，但从尖端到基部存在浓度梯度，这种分布模式多在花粉管发育的后期观察到。Rac蛋白在青杆花粉管不同发育时期的分布模式变化可能和花粉管的生长状态有关，在花粉管发育早期和中期，正是花粉管旺盛生长期，Rac蛋白的尖端定位保证了花粉管的极性生长。对Rac蛋白在花粉管的分布进行的连续切片扫描发现，Rac蛋白不但分布在质膜上，并与质膜偶联，而且在胞质中亦有分布。通过对一系列正常发育（即极性生长的花粉管）和畸形发育的花粉管进行观察发现，Rac蛋白主要分布在旺盛生长的花粉管尖端质膜或离顶端20 Vm处，在分叉的生长缓慢的分枝端分布较少。而在那些发生分叉生长的花粉管中，处于次要位置的基本停止生长的分枝端几乎没有Rac蛋白存在。在顶端发生膨大的花粉管中，Rac蛋白均匀分布在花粉管整个质膜上，丧失浓度梯度，失去极性生长。这些结果显示了Rac蛋白参与了青杆花粉管生长。 应用抗NtRacl抗体进行的间接免疫荧光显微镜定位实验，我们在正在生长的花粉管的管核中观察到明亮的荧光，显示了有Rac蛋白的存在。当精细胞在花粉粒中未移动到花粉管中时，几乎没有观察到荧光信号。随着花粉管发育，两个精细胞的位置发生变化，当其中一个较大的精细胞移动到花粉管中时，观察到明亮的荧光信号，这些结果显示了Rac蛋白可能参与了管核或精细胞在花粉管内的移动。

Morphine induces Beclin 1-and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus

Chronic exposure to morphine can induce drug addiction and neural injury, but the exact mechanism is not fully understood. Here we show that morphine induces autophagy in neuroblastoma SH-SY5Y cells and in the rat hippocampus. Pharmacological approach shows that this effect appears to be mediated by PTX-sensitive G protein-coupled receptors signaling cascade. Morphine increases Beclin 1 expression and reduces the interaction between Beclin 1 and Bcl-2, thus releasing Beclin 1 for its pro-autophagic activity. Bcl-2 overexpression inhibits morphine-induced autophagy, whereas knockdown of Beclin 1 or knockout of ATG5 prevents morphine-induced autophagy. In addition, chronic treatment with morphine induces cell death, which is increased by autophagy inhibition through Beclin 1 RNAi. Our data are the first to reveal that Beclin 1 and ATG5 play key roles in morphine-induced autophagy, which may contribute to morphine-induced neuronal injury.

Ultrafine PEG-PLA fibers loaded with both paclitaxel and doxorubicin hydrochloride and their in vitro cytotoxicity

By means of "emulsion-electrospinning", both hydrophobic and hydrophilic drugs, paclitaxel (PTX) and doxorubicin hydrochloride (DOX), were successfully loaded into PEG-PLA nanofiber mats to realize multi-drug delivery. The release behaviors of both the drugs from the same fiber mats were ascribed to their solubility properties and distribution status in the fibers. Due to its high hydrophilicity, DOX was easy to diffuse out from the fibers, and its release rate was always faster than that of hydrophobic PTX. Moreover, the release rate of PTX was accelerated by DOX's release from the same drug-loaded fibers. In vitro cytotoxicity against rat Glioma C6 cells indicated that the dual drug combination showed a higher inhibition and apoptosis against C6 cells than a single drug-loaded system, which suggests the promise for multi-drug delivery on combination therapy.

Triblock poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid)/paclitaxel conjugates: Synthesis, micellization, and cytotoxicity

A triblock poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) (PLA-PEG-PLA)/paclitaxel (PTX) conjugate was synthesized by the reaction of carboxyl-terminated copolymer PLA-PEG-PLA with PTX in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. Carboxyl-terminated copolymer PLA-PEG-PLA was prepared by the reaction of the hydroxyl end groups in copolymer PLA-PEG-PLA with succinic anhydride. Its structure was confirmed by NMR and gel permeation chromatography. The PLA-PEG-PLA/PTX conjugates could self-assemble into micelles in aqueous solutions with a low critical micelle concentration. Dynamic light scattering and environmental scanning electron microscopy analyses of the PLA-PEG-PLA/PTX micelles revealed their spherical structure and size of 220 nm. The antitumor activity of the conjugate against woman Hela cancer cells, evaluated by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide method, showed that the conjugates had an antitumor activity similar to that of pure PTX. The obtained PLA-PEG-PLA/PTX conjugates are expected to be used in clinical practice.

北大别黄土岭麻粒岩变质演化：对大别造山带演化进程的指示意义

Granulites from Huangtuling in the North Dabie metamorphic core complex in eastern China preserve rare mineralogical and mineral chemical evidence for multistage metamorphism related to Paleoproterozoic metamorphic processes, Triassic continental subduction-collision and Cretaceous collapse of the Dabieshan Orogen. Six stages of metamorphism are established, based on detailed mineralogical and petrological studies: (I) amphibolite facies (6.3–7.0 kbar, 520–550 °C); (II) high-pressure/high-temperature granulite facies (12–15.5 kbar, 920–980 °C); (III) cooling and decompression (4.8–6.0 kbar, 630-700 °C); (IV) medium-pressure granulite facies (7.7–9.0 kbar, 690–790 °C); (V) low-pressure/high-temperature granulite facies (4.0–4.7 kbar, 860–920 °C); (VI) retrograde greenschist facies overprint (1–2 kbar, 340–370 °C). The P–T history derived in this study and existing geochronological data indicate that the Huangtuling granulite records two cycles of orogenic crustal thickening events. The earlier three stages of metamorphism define a clockwise P–T path, implying crustal thickening and thinning events, possibly related to the assembly and breakup of the Columbia Supercontinent ca. 2000 Ma. Stage IV metamorphism indicates another crustal thickening event, which is attributed to the Triassic subduction/collision between the Yangtze and Sino–Korean Cratons. The dry lower crustal granulite persisted metastable during the Triassic subduction/collision due to lack of hydrous fluid and deformation. Stage V metamorphism records the Cretaceous collapse of the Dabieshan Orogen，possibly due to asthenosphere upwelling or removal of the lithospheric mantle resulting in heating of the granulite and partial melting of the North Dabie metamorphic core complex. Comparison of the Huangtuling granulite in North Dabie and the high-pressure (HP)–ultrahigh-pressure (UHP) metamorphic rocks in South Dabie indicates that the subducted upper (South Dabie) and lower (North Dabie) continental crusts underwent contrasting tectonometamorphic evolution during continental subduction–collision and orogenic collapse. High-pressure granulites are generally characterized by the absence of orthopyroxene. However, the Huangtuling felsic granulite rarely preserves the high-pressure granulite facies assemblage of garnet + orthopyroxene + biotite + plagioclase + K-feldspar + quartz. To investigate the effects of bulk rock composition on the stability of orthopyroxene-bearing, high-pressure granulite facies assemblages in the NCKFMASHTO (Na2O–CaO–K2O–FeO–MgO–Al2O3–SiO2–H2O–TiO2–Fe2O3) system, we constructed a series of P–T–X pseudosections based on the melt-reintegrated composition of the Huangtuling felsic high-pressure granulite. Our calculations demonstrate that the orthopyroxene-bearing, high-pressure granulite facies assemblages are restricted to low XAl [Al2O3/(Na2O + CaO + K2O + FeO + MgO + Al2O3) < 0.35, mole proportion] or high XMg [MgO/(MgO + FeO) > 0.85] felsic–metapelitic rock types. Our study also reveals that the XAl values in the residual felsic–metapelitic, high-pressure granulites could be significantly reduced by a high proportion of melt loss. We suggest that orthopyroxene-bearing high-pressure granulites occur in residual overthickened crustal basement under continental subduction–collision zones and arc–continent collision belts.

富磷过铝质岩浆体系的实验研究

富磷过铝质岩浆是过铝质岩浆体系中的一个重要类型，它通常以富P，高ASI[铝饱和指数，Al2O3/(Na2O+K2O+CaO)摩尔比值]，贫Fe、Mg、Ca，强烈亏损REE、Th、Y，与稀有金属(W、Sn、Be、Nb、Ta)矿化具有成因联系为特征。富磷过铝质岩浆体系的性状及其演化的地球化学特征、岩浆液态分离、磷酸盐-硅酸盐矿物对平衡对熔体相中磷的制约以及富磷过铝质岩浆－热液体系中微量元素(包括REE)地球化学行为等科学问题尚未得到系统解决，目前更缺乏实验地球化学的直接证据。对上述科学问题的研究、探索，将有助于了解富磷过铝质岩浆体系形成和演化的地球化学特征，有助于揭示磷对微量元素(包括稀有、稀土元素)地球化学行为的影响，这对于理解过铝质岩浆体系成岩、成矿作用过程具有重要的理论和实际意义。 本文以天然钠长花岗岩为初始物，针对上述内容，开展了不同PTX条件下高温高压下实验研究，获得了如下重要认识： 1) 确定了100 MPa条件下磷对过铝质岩浆液相线温度的影响。随着体系中P2O5的含量增大，液相线温度由1.91 wt%P2O5的780 C降至4.83 wt%P2O5的760C、7.71 wt%P2O5的740 C，即体系中每增加1 wt%P2O5，液相线温度降低约7~10 C； 2) 不同PTX条件下的实验产物中均未见不混溶球粒结构、乳滴结构或流动构造，初步推断富磷过铝质岩浆体系中可能不存在单纯由磷引起的岩浆液态分离现象； 3) 锰铝榴石-磷灰石矿物平衡反应制约着形成过铝质初始岩浆中P2O5含量，熔体相中P2O5含量在750 C的0.47~0.80 wt%、830 C的0.35~2.26 wt%范围内。熔体相中的P2O5含量与ASI之间存在二次函数关系(P2O5 wt%=3.5×ASI2-11.3×ASI+9.5 )。锰铝榴石溶解，促使体系中Al2O3活度增大，含锰氟磷灰石的溶解及其端元磷灰石结晶之间的化学平衡，导致熔体相中P2O5的降低，很可能是熔体中P2O5随体系ASI增大而降低的机制。 4) 不同PTX条件下稀有金属元素(W、Sn、Be、Nb、Ta)在流体/熔体相间的分配系数(Dif/m)<<0.1，预示着W、Sn、Be、Nb、Ta强烈富集在富磷过铝质熔体相中。由此，可推测富P过铝质岩浆体系的演化晚期，不太可能分异出富含上述成矿元素的成矿流体。随着岩浆分异演化的进行，残余熔体相中最终导致绿柱石、锆石、锡石、铌钽矿物等矿物饱和结晶，形成有经济意义的花岗岩型或伟晶岩型稀有金属矿床。 5) 不同PTX条件下REE在流体/熔体间的分配系数(DREE)随REE的原子序数增大而逐渐降低，构成右倾的平滑曲线，不存在在Nd-Pm、Gd、Ho-Er处的拐点；Y与Ho在流体/熔体相间分配系数的比值(DY/DHo)约为1，不受体系温度、压力和P2O5含量变化的影响。上述实验结果揭示，富磷过铝质量岩浆演化至岩浆-热液过渡阶段，熔体-流体作用不会导致Y-Ho间的分异，不会引起REE间的分异，因此，富磷过铝质岩浆演化晚期的熔体-流体作用过程不可能是产生稀土“四分组”效应的根本机理。