低氧预适应对离体星形胶质细胞低氧耐受性的影响

目的：研究低氧预适应对体外培养的星形胶质细胞低氧耐受性的影响。方法：体外培养的鼠脑星形胶质细胞,随机分为对照组（control,C组）,低氧损伤组（hypoxia,H组）,低氧预适应组（hypoxic preconditioning,HP组）,通过检测细胞MTT代谢变化、凋亡发生和形态学观察探讨低氧预适应对星型胶质细胞低氧损伤的保护作用;免疫细胞化学方法分析Bcl-2和Bax的表达差异。结果：与低氧组相比,HP48、HP72组MTT代谢活性较高。免疫细胞化学结果提示低氧预适应组Bcl-2表达高于低氧损伤组,低氧预适应组Bax表达低于低氧损伤组。结论：低氧预适应对大鼠星形胶质细胞低氧损伤有保护作用,可能与Bax表达受抑,维持Bcl-2表达有关,通过对抗凋亡程序的发展产生保护作用。     

低氧预处理对新生大鼠脑低氧缺血时海马区Bcl-2和Bax表达的影响

目的：观察低氧预处理对新生大鼠脑低氧缺血时海马区Bcl-2和Bax表达的影响,探讨低氧预处理对新生大鼠脑低氧缺血损伤的保护机制。方法：7日龄新生SD大鼠随机分为正常对照组、假手术组、低氧缺血组（HIBD组）和低氧预处理组（HPC＋HIBD组）。采用免疫组织化学方法,检测各组脑组织海马区Bcl-2和Bax表达的变化。结果：与正常对照组、假手术组相比．HIBD组和HPC＋HIBD组海马区Bcl-2蛋白和Bax蛋白表达明显增多;与HIBD组相比,HPC＋HIBD组海马区Bcl-2蛋白表达明显增多,Bax蛋白表达明显减少。结论：低氧预处理后Bcl-2表达上调,Bax表达下调,可能是其保护随后脑低氧缺血损伤的机制之一。     

大鼠侧脑室注射腺苷A1受体反义寡聚脱氧核苷酸抑制脑缺血耐受的诱导

目的:观察侧脑室注射腺苷A1受体(ARA1)反义寡聚脱氧核苷酸(As-ODN)对脑缺血预处理(CIP)脑保护作用的影响,进一步探讨腺苷A1受体在CIP脑保护作用中的作用。方法:将54只凝闭双侧椎动脉的Wistar大鼠分为Sham组、CIP组、损伤性脑缺血组、CIP 损伤性脑缺血组、双蒸水 CIP 损伤性脑缺血组、ARA1As-ODN组、ARA1As-ODN CIP组、和ARA1As-ODN CIP 损伤性脑缺血组。ARA1As-ODN的剂量分为10nmol/5μl和20nmol/5μl,溶于双蒸水中,侧脑室注射。所有动物均在Sham手术后或末次全脑缺血/再灌注后7d断头取脑,硫堇染色观察海马CA1区锥体神经元迟发性死亡(DND)情况。结果:Sham组和CIP组均未见DND。与Sham、CIP组相比,损伤性脑缺血组出现了明显的DND,表现为组织学分级(HG)升高和锥体神经元密度(ND)下降(P<0.05)。CIP可显著抑制损伤性脑缺血引起的DND。与CIP 损伤性缺血组相比,ARA1As-ODN CIP 损伤性脑缺血组出现了显著的DND,表现为HG升高、ND降低(P<0.05),这种变化与ARA1As-ODN的剂量呈明显正相关。结论:腺苷A1受体As-ODN可阻断CIP诱导的脑缺血耐受,进一步证实了腺苷A1受体表达上调参与CIP诱导的脑缺血耐受。     

丹参酮ⅡA与丹皮酚配伍对大鼠脑缺血再灌注损伤保护作用研究

目的：研究丹参酮ⅡA与丹皮酚配伍（简称双丹配伍）对大鼠局灶性脑缺血再灌注损伤的脑梗死体积、自由基变化的影响,探讨双丹配伍对脑缺血损伤的保护作用。方法：复制大鼠中动脉缺血再灌注模型,分别给予双丹配伍干预,观察和评价受试动物行为学、脑梗死率、脑指数、脑含水量、SOD、MAD等指标的变化。结果：①双丹配伍的各试药组均具有明显改善局灶性脑缺血再灌注损伤大鼠的神经行为学评分,降低脑梗死率、脑指数、脑含水量、提高脑组织SOD活性、降低MDA含量;②双丹配伍各组中1：3配伍组的总体药效作用优于1：2和1：4组,但数据未见统计学差异;⑧双丹配伍尾静脉注射的药效作用明显优于预先灌胃组。结论：双丹配伍脂质体给大鼠口服和注射对脑缺血再灌注损伤具有显著保护作用。     

银杏内酯B和低氧预适应对小鼠急性缺氧损伤的保护作用

目的：通过观察缺氧预适应和银杏内酯B预处理对小鼠急性缺氧的影响,了解银杏内酯B的脑保护作用。方法：采用小鼠常压缺氧模型,观察小鼠的行为学并记录各组小鼠的最后死亡时间,脑组织含水量,用RT-PCR、Western blot分别检测各组小鼠皮层组织中RTP801mRNA表达和EPO蛋白表达。结果：银杏内酯B和低氧预适应均能明显延长常压缺氧小鼠的存活时间,降低脑水肿程度,并且银杏内酯组和低氧预适应组RTP801mRNA表达和EPO的表达均明显增加。结论：银杏内酯B与低氧预适应具有相类似的对抗小鼠急性低氧的作用,其脑保护作用与上调RTPS01mRNA和EPO蛋白的表达有关。     

低氧调节大鼠前额叶皮层IGFs家族基因的表达

目的：探讨急性和慢性低氧对胰岛素样生长因子（IGFs）家族中IGF-I,IGF一Ⅱ,IGF-1R和IGFBPlmRNA表达变化的调节。方法：我们利用低压低氧舱模拟5km低氧环境,研究大鼠暴露于急性和慢性低氧后的前额叶皮层IGF家族基因的表达变化。结果：急性和慢性连续低氧暴露后,大鼠前额叶皮层中IGF-I,IGF-II,IGF-1R和IGFBPlmRNA对低氧应激表现出明显不同的变化模式。急性低氧时,IGF-I和IGFBPlmRNA表达显著升高。而在慢性连续低氧暴露5～15d后,IGF-I和IGFBPlmRNA表达量逐渐回复到对照水平。但IGF-1R的表达仍然保持较高水平。结论：急性低氧上调IGF基因表达可能参与皮层神经元的保护,而慢性低氧升高的IGF-1R基因表达可能参与慢性低氧损伤的适应过程。     

促红细胞生成素对大鼠局灶性脑缺血再灌注神经元的保护作用及P53蛋白的表达

目的:探讨促红细胞生成素(Epo)对大鼠局灶性脑缺血再灌注神经细胞的保护作用.方法:60只SD大鼠随机分为缺血再灌注Epo治疗组(又分为高剂量A组、低剂量B组)、缺血再灌注组(C组)及假手术组(D组),采用大脑中动脉线栓法制备大鼠局灶性脑缺血再灌注模型.参考Longa的5分制法在大鼠麻醉清醒后进行评分,TTC染色法观察线栓侧的梗死体积,并检测脑组织含水量的变化,HE染色法观察脑缺血再灌注后脑组织的病理变化,TUNEL法观察神经细胞凋亡情况,western blot法观察p53蛋白的表达变化.结果:对照组比较,大鼠脑缺血再灌注后出现不同程度的脑梗死,24h后缺血中心区及周围区均可见到p53蛋白表达.缺血再灌注6h内给予Epo可显著改善大鼠神经功能评分,减少梗死体积及脑组织含水量,减轻病理学变化及神经细胞凋亡.结论:Epo通过调控神经细胞凋亡、改善缺血再灌注损伤而发挥脑保护作用,P53蛋白参与缺血再灌注后神经细胞凋亡机制.     

孕酮对新生大鼠低氧缺血性脑损伤中MMP-3表达的影响

目的：研究孕酮（PROG）对新生大鼠低氧缺血后脑内基质金属蛋白酶3（MMP-3）表达的影响。方法：建立新生大鼠低氧缺血性脑损伤动物模型,伊文思兰（EB）染色和电镜观察新生鼠低氧缺血性脑损伤血一脑屏障的通透性改变;免疫印迹（Western blot）方法检测大脑皮层MMP-3表达。结果：电镜显示低氧缺血组血-脑屏障完整性明显破坏：EB染色结果表明低氧缺血组血-脑屏障通透性明显高于假手术组,差异极显著（P〈0．01）,孕酮组血-脑屏障通透性明显低于低氧缺血组,有显著性差异（P〈0．05）;Western blot结果显示低氧缺血组MMP-3蛋白表达显著高于假手术组（P〈0．01）;孕酮组MMP-3蛋白表达显著低于低氧缺血组（P〈0．05）。结论：孕酮通过减少MMP-3的表达,降低血一脑屏障的损伤,这可能是其发挥脑保护作用的机制之一。     

低氧大鼠脑线粒体体外转录活性的研究

目的：探讨低氧对大鼠脑线粒体DNA表达的影响及其与能量生成的关系。方法：雄性Wistar大鼠随机分为3组：急性低氧组（AH）、慢性低氧组（CH）和对照组,其中急、慢性低氧组动物分别连续暴露于模拟海拔4000m高原3d(AH)和40d（CH）。分离脑线粒体,分别测定线粒体体外转录活性、F0F1－ATP酶活性以及ATP对线粒体体外转录的影响。结果：急性低氧大鼠脑线粒体体外转录活性及F0F1－ATP酶活性显著降低,慢性低氧时有所回升,两者呈线性相关。ATP对大鼠脑线粒体体外转录活性呈双相效应。结论：低氧时脑线粒体转录活性改变可能参与低氧抑制线粒体能量代谢的机制,ATP可能通过反馈作用对线粒体转录进行微调。     

硬膜外皮质电刺激治疗急性颅脑脑损伤的初步实验研究

目的:探讨硬膜外皮质电刺激(EECS)对急性颅脑脑损伤(ACCI)后的影响.方法:SD大鼠24只,随机分为A组(假损伤组),B组(损伤组)和C组(治疗组),侧方液压打击损伤(LFPI)大鼠脑组织建立ACCI模型后,C组给予硬膜外电刺激治疗.术后24小时处死动物,检测A组脑组织含水量、血清神经元特异性烯醇化酶(NSE)含量与B、C组上述指标的差异.结果:与A组相比,B组与C组脑组织含水量明显增加(P＜0.01),与B组相比,C组大鼠脑含水量减少(P＜0.05),差异有统计学意义;与A组相比.B组与C组大鼠血清NSE明显增加(P＜0.01),与B相比,C组大鼠大鼠血清NSE明显减少(P＜0.01),差异有统计学意义.结论:EECS可减轻ACCI后脑水肿,降低血清NSE水平,从而起到脑保护的作用.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

How does the host-specialized aphid deal with food deficiency？

Aphis gossypii Glover shows obvious host specialization, with cucurbit- and cotton-specialized biotypes or host races in many regions. Because its annual natal hostcrops senesce earlier the cucurbit-specialized biotype may suffer food deficiency. The method this biotype uses to overcome this challenge is still poorly understood. In orderto understand the potential of the cucurbit-specialized biotype aphids in host shift and usage, the performance of this biotype on cotton （Gossypium hirsutum）, a common butpoor quality host plant, was explored in this study. The cucurbit-specialized aphids could establish populations on cotton only when these plants had at least nine leaves, and subsequent populations developed rather slowly. The presence of whitefly populations on cotton improved the success rate of cucurbit-specialized aphids. The cucurbit-specialized aphidswere mainly distributed on the older leaves of cotton, with only a few settling on the upper leaves. The cucurbit-specialized aphids reared on cotton for 40, 54 and 61 days stillmaintained strong preference for their natal host plant, cucumber （Cucumis sativus）, rather than cotton, and their net reproductive rates and intrinsic rates of natural increase weredramatically lower when they were transferred onto new six-leaf cotton plants or detached leaves. Therefore, we concluded that the cucurbit-specialized aphids have the potentialto utilize mature or whitefly-stressed cotton plants, but that this feeding experience on cotton did not alter their specialization for cucurbits. Some cotton plants could act as atemporary host for the cucurbit-specialized aphids to overcome food deficiency arising from senescing cucurbits.     

Plastid Signals and the Bundle Sheath： Mesophyll Development in Reticulate Mutants

The development of a plant leaf is a meticulously orchestrated sequence of events producing a complex organ comprising diverse cell types. The reticulate class of leaf variegation mutants displays contrasting pigmentation between veins and interveinal regions due to specific aberrations in the development of mesophyll cells. Thus, the reticulate mutants offer a potent tool to investigate cell-type-specific developmental processes. The discovery that most mutants are affected in plastid-localized, metabolic pathways that are strongly expressed in vasculature-associated tis- sues implicates a crucial role for the bundle sheath and their chloroplasts in proper development of the mesophyll cells. Here, we review the reticulate mutants and their phenotypic characteristics, with a focus on those in Arabidopsis thali- ana. Two alternative models have been put forward to explain the relationship between plastid metabolism and meso- phyll cell development, which we call here the supply and the signaling hypotheses. We critically assess these proposed models and discuss their implications for leaf development and bundle sheath function in C3 species. The characteriza- tion of the reticulate mutants supports the significance of plastid retrograde signaling in cell development and highlights the significance of the bundle sheath in C3 photosynthesis.     

Knights in Action： Lectin Receptor-Like Kinases in Plant Development and Stress Responses

The Receptor-Like Kinase （RLK） is a vast protein family with over 600 genes in Arabidopsis and 1100 in rice. The Lectin RLK （LecRLK） family is believed to play crucial roles in saccharide signaling as well as stress perception. All the LecRLKs possess three domains： an N-terminal lectin domain, an intermediate transmembrane domain, and a C-terminal kinase domain. On the basis of lectin domain variability, LecRLKs have been subgrouped into three subclasses： L-, G-, and C-type LecRLKs. While the previous studies on LecRLKs were dedicated to classification, comparative structural analysis and expression analysis by promoter-based studies, most of the recent studies on LecRLKs have laid special emphasis on the potential of this gene family in regulating biotic/abiotic stress and developmental pathways in plants, thus mak- ing the prospects of studying the LecRLK-mediated regulatory mechanism exceptionally promising. In this review, we have described in detail the LecRLK gene family with respect to a historical, evolutionary, and structural point of view. Furthermore, we have laid emphasis on the LecRLKs roles in development, stress conditions, and hormonal response. We have also discussed the exciting research prospects offered by the current knowledge on the LecRLK gene family. The multitude of the LecRLK gene family members and their functional diversity mark these genes as both interesting and worthy candidates for further analysis, especially in the field of crop improvement.     

Aphis （Hemiptera： Aphididae） species groups found in the Midwestern United States and their contribution to the phylogenetic knowledge of the genus

A phylogeny of the genus Aphis Linnaeus, 1 758 was built primarily from specimens collected in the Midwest of the United States. A data matrix was constructedwith 68 species and 41 morphological characters with respective character states of alate and apterous viviparous females. Dendrogram topologies of analyses performed usingUPGMA （Unweighted Pair Group Method with Arithmetic Mean）, Maximum Parsimony and Bayesian analysis of Cytochrome Oxidase I, Elongation Factor 1-α and primary endosymbiont Buchnera aphidicola 16S sequences were not congruent. Bayesian analysis strongly supported most terminal nodes of the phylogenetic trees. The phylogeny wasstrongly supported by EFI-α, and analysis of COl and EFI-α molecular data combined with morphological characters. It was not supported by single analysis of COI or Buch-hera aphidicola 16S. Results from the Bayesian phylogeny show 4 main species groups： asclepiadis,fabae, gossypii, and middletonii. Results place Aphis and species of the generaProtaphis Bōrner, 1952, Toxoptera Koch, 1856 and Xerobion Nevsky, 1928 in a monophyletic clade. Morphological characters support this monophyly as well. The phylogenyshows that the monophyletic clade of the North American middletonii species group belong to the genus Protaphis： P. debilicornis （Gillette ＆ Palmer, 1929）, comb. nov., P. echinaceae（Lagos and Voegtlin, 2009）, comb. nov., and P. middletonii （Thomas, 1879）. The genus Toxoptera should be considered a subgenus of Aphis （stat. nov.）. The analysis also indicatesthat the current genus Iowana Frison, 1954 should be considered a subgenus of Aphis （stat. nov.）.     

Open and Closed： The Roles of Linker Histones in Plants and Animals

Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications （PTMs） to core histones, but linker histone function is less well understood. In this review, we describe the current understand- ing of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and devel- opment, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.