Identification of two novel type 1 peroxisomal targeting signals in Arabidopsis thaliana

Peroxisomes lack their own genetic material and must therefore import proteins encoded by genes in the nucleus. Amino acids within these proteins serve as targeting signals: they direct the delivery of the proteins to the organelle. The majority of soluble proteins destined for the peroxisomal matrix utilize a type 1 peroxisomal targeting signal (PTS1): a C-terminal tripeptide that follows the pattern small/basic/hydrophobic. We have discovered two new C-terminal tripeptides that target proteins to peroxisomes in Arabidopsis thaliana. The tripeptides PSL and KRR do not fit the major PTS1 consensus but cause green fluorescent protein to accumulate in peroxisomes of stably transformed Arabidopsis. We have identified forty-one proteins in the Arabidopsis genome that also bear these tripeptides at their C-termini and may therefore be peroxisomal.     

Photochemically Mediated Atom Transfer Radical Polymerization Using Polymeric Semiconductor Mesoporous Graphitic Carbon Nitride

Photochemically mediated atom transfer radical polymerization of vinyl monomers is successfully activated by ecofriendly heterogeneous mesoporous graphitic carbon nitride (mpg‐C₃N₄). This method pertains to the use of mpg‐C₃N₄ as photoactivator for reduction of initially loaded copper(II) species, thus promoting the in situ formation of the copper(I) species. The controlled nature of the polymerizations in both natural sunlight and UV‐light irradiation at ambient temperature is confirmed by the good agreement of the kinetics of the ­polymerization with theoretical values. The light on–off experiments ­demonstrate that polymerizations are clearly initiated and moderated by either UV light or sunlight.

     

白色假丝酵母菌pACT1-GFP质粒的构建及表达

目的构建能够稳定表达绿色荧光蛋白（GFP）的白色假丝酵母菌菌株,以便对目的基因进行示踪。方法构建pACT1-GFP质粒,以白色假丝酵母菌CAI4菌株作为感受态进行转化培养后,分别观察绿色荧光蛋白在两相型下的表达情况。结果含有pACT1-GFP的白色假丝酵母菌菌株,99%在两相型下均有较高水平的荧光蛋白表达,且荧光强度没有明显差别。结论pACT1-GFP能够在白色假丝酵母菌体内稳定表达。     

吲哚菁绿血管造影在颅内外搭桥手术中的应用

目的探讨吲哚菁绿（ICG）血管造影在颅内外血管搭桥术中判定吻合口通畅性方面的作用。方法回顾性分析2009年12月至2012年12月57例患者的临床资料,共行颅内外搭桥手术61例次,共68处吻合口。吻合术后均行ICG血管造影,观察搭桥血管通畅情况,并与术后1周内的数字减影血管造影（DSA）或CT血管造影（CTA）检查结果比较。结果术中ICG血管造影有良好的时间和空间分辨率,发现4处吻合口闭塞,给予重新吻合。最终造影显示67处通畅,1处闭塞。术后DSA和CTA检查示66处吻合口通畅,2处闭塞。结论术中ICG血管造影可以判断颅内外搭桥吻合口的通畅性,是一种简便、迅速、准确的术中血管造影技术。     

慢病毒载体感染人口腔黏膜成纤维细胞稳定表达绿色荧光蛋白的研究

目的：确立慢病毒载体高效感染人口腔黏膜成纤维细胞的感染条件,为进一步应用基因工程技术研究人口腔黏膜成纤维细胞奠定基础。方法：采用组织块法培养原代人口腔黏膜成纤维细胞,免疫组化SP法鉴定细胞类型,将携带绿色荧光蛋白基因的慢病毒载体以不同感染复数（multiplicity of infection,MOI）感染纯化细胞,并设置不同感染条件,感染4 d后于荧光显微镜下观察绿色荧光蛋白表达情况。结果：成功分离纯化得到人口腔黏膜成纤维细胞,当慢病毒感染复数为30,polybrene浓度为8μg/mL,并加入感染增强液时,可达到较高的感染效率,满足后续实验需要。结论：慢病毒载体可高效感染人口腔黏膜成纤维细胞,携带的绿色荧光蛋白基因可在成纤维细胞内稳定表达。     

吲哚菁绿清除试验对急性/慢加急性肝衰竭患者预后的评价

目的通过观察肝衰竭患者吲哚菁绿清除试验15 min滞留率(ICG R15)与其他常用肝脏储备功能指标变化,评判ICGR15对预测肝衰竭患者预后的价值。方法对32例肝衰竭患者采用脉搏光度分析法(即PDD法),检测入院时ICG R15,同时检测常用肝功能指标,包括ALT、AST、TBil、Alb、GGT、胆碱酯酶(CHE)、凝血酶原活动度(PTA)和胆固醇(CHO)。计算MELD评分。同时选取15例慢性肝炎患者、94例肝硬化患者作为对照,检测ICG R15及同期Alb、CHE、PTA和CHO。对肝衰竭组患者进行了3个月随访,确定存活及死亡情况,并对ICG R15、MELD评分进行受试者工作特征曲线(ROC曲线)分析,探讨其对预后的判断意义。结果(1)在慢性肝炎、肝硬化和肝衰竭组ICG R15、Alb、PTA、CHE、CHO在3组间差异均有统计学意义(P<0.05),其中ICG R15,Alb,CHE,PTA在慢性肝炎和肝硬化组差异有统计学意义(P<0.05);ICG R15、PTA、CHO在肝硬化和肝衰竭组差异有统计学意义(P<0.05)。(2)3个月时肝衰竭存活组与死亡组ICG R15分别为(50.05±9.04)%、(56.27±5.65)%,差异有统计学意义(P<0.05)。(3)对肝衰竭组ICG R15和MELD评分进行ROC曲线分析,二者曲线下面积分别为75.9%和60.4%,ICG R15对预后的判断优于MELD评分;当ICG R15为52.5%时对预后判断的敏感性为80%,特异性为70.6%。(4)肝衰竭组入院时ICG R15≤50%10例,3个月时死亡2例,病死率20%;>50%22例,3个月时死亡14例,病死率63.6%。结论 (1)在慢性肝炎、肝硬化和肝衰竭组,随着病情加重PTA逐渐下降,ICG R15逐渐升高,两者反映病情的严重程度优于其他指标。(2)比较ICG R15和MELD评分对肝衰竭患者的预后判别作用,ICG R15对3个月预后的判断优于MELD评分。(3)肝衰竭患者ICG R15>50%时预后较差。     

Cavernous antioxidant effect of green tea,epigallocatechin‐3‐gallate with/without sildenafil citrate intake in aged diabetic rats

This study aimed to assess the cavernous antioxidant effect of green tea (GT), epigallocatechin‐3‐gallate (EGCG) with/without sildenafil citrate intake in aged diabetic rats. One hundred and four aged male white albino rat were divided into controls that received ordinary chow, streptozotocin (STZ)‐induced aged diabetic rats, STZ‐induced diabetic rats on infused green tea, induced diabetic rats on epigallocatechin‐3‐gallate and STZ‐induced diabetic rats on sildenafil citrate added to EGCG. After 8 weeks, dissected cavernous tissues were assessed for gene expression of eNOS, cavernous malondialdehyde (MDA), glutathione peroxidase (GPx), cyclic guanosine monophosphate (cGMP), and serum testosterone (T). STZ‐induced diabetic rats on GT demonstrated significant increase in cavernous eNOS, cGMP, GPx and significant decrease in cavernous MDA compared with diabetic rats. Diabetic rats on EGCG demonstrated significant increase in cavernous eNOS, cGMP, GPx and significant decrease in cavernous MDA compared with diabetic rats or diabetic rats on GT. Diabetic rats on EGCG added to sildenafil showed significant increase in cavernous eNOS, cGMP and significant decrease in cavernous MDA compared with other groups. Serum T demonstrated nonsignificant difference between the investigated groups. It is concluded that GT and EGCG have significant cavernous antioxidant effects that are increased if sildenafil is added.     

Distribution and intrinsic membrane properties of basal forebrain GABAergic and parvalbumin neurons in the mouse

The basal forebrain (BF) strongly regulates cortical activation, sleep homeostasis, and attention. Many BF neurons involved in these processes are GABAergic, including a subpopulation of projection neurons containing the calcium‐binding protein, parvalbumin (PV). However, technical difficulties in identification have prevented a precise mapping of the distribution of GABAergic and GABA/PV+ neurons in the mouse or a determination of their intrinsic membrane properties. Here we used mice expressing fluorescent proteins in GABAergic (GAD67‐GFP knock‐in mice) or PV+ neurons (PV‐Tomato mice) to study these neurons. Immunohistochemical staining for GABA in GAD67‐GFP mice confirmed that GFP selectively labeled BF GABAergic neurons. GFP+ neurons and fibers were distributed throughout the BF, with the highest density in the magnocellular preoptic area (MCPO). Immunohistochemistry for PV indicated that the majority of PV+ neurons in the BF were large (>20 μm) or medium‐sized (15–20 μm) GFP+ neurons. Most medium and large‐sized BF GFP+ neurons, including those retrogradely labeled from the neocortex, were fast‐firing and spontaneously active in vitro. They exhibited prominent hyperpolarization‐activated inward currents and subthreshold “spikelets,” suggestive of electrical coupling. PV+ neurons recorded in PV‐Tomato mice had similar properties but had significantly narrower action potentials and a higher maximal firing frequency. Another population of smaller GFP+ neurons had properties similar to striatal projection neurons. The fast firing and electrical coupling of BF GABA/PV+ neurons, together with their projections to cortical interneurons and the thalamic reticular nucleus, suggest a strong and synchronous control of the neocortical fast rhythms typical of wakefulness and REM sleep. J. Comp. Neurol., 521:1225–1250, 2013. © 2012 Wiley Periodicals, Inc.     

三种方法浓缩慢病毒后绿色荧光蛋白转染效率的比较

 目的 比较超滤管法、超速离心法及病毒浓缩液法浓缩慢病毒后绿色荧光蛋白(green fluorescent protein,GFP)的转染效率。方法 用摇床过夜摇pMD2.G、psPAX2及GFP载体菌种并用试剂盒提取这三种质粒;将pMD2.G、psPAX2及GFP载体质粒与转染试剂按比例混合后转染293T细胞生产病毒;收集病毒上清液并用超滤管法、超速离心法及病毒浓缩液法浓缩慢病毒;将接种的293T细胞分为3组,并向每组接种有293T细胞的培养液中加入2、4、8、16 μl三种方法浓缩的病毒浓缩液,48 h后用荧光显微镜观察和流式细胞仪检测三种方法浓缩慢病毒后转染293T细胞的GFP表达情况。结果 荧光显微镜观察结果显示随着转染量的增加,GFP表达率增加,超滤管法浓缩病毒转染293T细胞后GFP的表达效率最高;流式检测显示病毒浓缩液法组2、4、8、16 μl GFP的表达率分别为(2.4±0.1)%、(3.8±0.3)%、(8.2±0.6)%、(12.2±0.3)%;超滤管法组2、4、8、16 μl GFP的表达率分别为(5.4±0.3)%、(7.4±0.4)%、(12.7±0.1)%、(17.3±0.6)%;超速离心法组2、4、8、16 μl GFP的表达率分别为(0.7±0.1)%、(1.2±0.1)%、(2.1±0.1)%、(0.4±0.2)%。超滤管法组与其他两组比较,P<0.05,差异有统计学意义。结论 不同方法浓缩慢病毒后的GFP转染效率不同,三种慢病毒浓缩方法中超滤管法浓缩慢病毒后的GFP转染效率最高。