东亚钳蝎氯毒素通过下调PKM2介导的有氧糖酵解抑制神经胶质瘤细胞的增殖活性

东亚钳蝎氯离子通道毒素(Buthus martensii Karshch chlorion channel toxin,BmK CT)是一类短链神经毒素,在体内和体外均能有效抑制神经胶质瘤细胞侵袭和增殖。然而,BmK CT抑制胶质瘤细胞增殖活性的机制尚不清楚。本研究采用代谢组学的方法探索BmK CT抑制人脑神经胶质瘤细胞增殖的分子机制。首先,通过蛋白质表达纯化获得带有谷胱甘肽S转移酶(glutathione S-transferase, GST)标签的BmK CT蛋白和GST蛋白。利用圆二色谱检测两种蛋白质均具有α-螺旋二级结构,并且融合蛋白质GST-BmK CT热稳定性较好。通过噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide, MTT)法检测GST-BmK CT对神经胶质瘤细胞(U251和A172)增殖的影响。结果显示,与GST处理组相比,1μmol/L GST-BmK CT处理组对U251细胞的抑制率更明显(U251细胞19±1.1 vs. 2.25±0.95,P<0.001, 48 h; A172细胞12±1.4 vs. 2.25±1.25,P<0.001, 48 h)。利用气相色谱-质谱联用仪(gas chromatography-mass spectrometry, GC-MS)分析鉴定出细胞内的63种代谢物,通过通路分析和聚类分析证实,GST-BmK CT处理影响神经胶质瘤细胞的有氧糖酵解。根据峰面积统计数据显示,GST-BmK CT处理降低胞内丙酮酸的含量。GST-BmK CT处理组相比GST组,明显下调胞外培养基的乳酸分泌(16.33±3.78 vs. 35.6±2.31,P<0.001, 48 h),葡萄糖消耗(2.04±0.09 vs. 2.64±0.04,P<0.05, 48 h)和胞内腺苷三磷酸(ATP)含量(633.79±0.001 vs. 5392.71±266.67,P<0.05, 48 h)。结果表明,GST-BmK CT通过下调胶质瘤细胞有氧糖酵解水平降低ATP的生成。实时荧光定量PCR和蛋白质免疫印迹结果显示,GST-BmK CT降低U251细胞丙酮酸激酶(pyruvate kinase M2, PKM2)在转录水平和翻译水平的表达,进一步发现GST-BmK CT通过下调低氧诱导因子(hypoxia inducible factor-1α, HIF-1α)降低PKM2在转录水平的表达。以上结果表明,BmK CT通过下调HIF-1α的表达,降低PKM2介导的有氧糖酵解从而抑制神经胶质瘤细胞的增殖活性。     

水温对刀鲚幼鱼耗氧率、窒息点、血糖及肌肝糖元指标的影响

实验测定了不同水温条件下刀鲚(Coilia nasus)幼鱼的耗氧率和窒息点,并对耗氧率的昼夜变化规律以及水温对刀鲚幼鱼血糖及肌肝糖元指标的影响进行了研究,旨在为刀鲚运输、养殖生产中制定合理的放养密度和养殖工艺提供参考依据.结果表明:在试验水温(16℃～28C)条件下,刀鲚幼鱼的耗氧率随水温的升高而增大,耗氧率(R)与水温(T)的回归方程为R=0.988T2-32.36T+343.7,相关系数为0.96;刀鲚幼鱼的耗氧率具有昼夜节律性,耗氧高峰出现在7:00,低谷在21:00;刀鲚幼鱼的窒息点与水温显著相关,窒息点A(mg·L-1)与水温T(℃)的回归关系为A=0.9397T0.8001,R2=0.98.刀鲚幼鱼血清中血糖含量在水温16℃～24℃阶段保持相对稳定,血糖含量在17.24 ～ 19.79 mmol·L-1,肌肉和肝脏糖元含量均呈波浪式变动;但在水温24℃～28℃阶段,血糖含量随水温上升而显著升高,而肌肉和肝脏糖元含量均显著降低.     

南方红豆杉人工引种及野生植株活性成分含量与分布

南方红豆杉是我国特有的濒危植物,因其具有抗癌药用价值而被过度采集,导致现存种群减少.采用超声辅助提取和HPLC检测人工引种及野生南方红豆杉植株中紫杉醇、7-木糖紫杉醇、三尖杉宁碱等活性成分含量,分析其在不同部位、年限和坡向的分布规律.结果表明:南方红豆杉人工引种后,植株中的紫杉醇、7-木糖紫杉醇和三尖杉宁碱含量变化明显,野生植株中紫杉醇含量显著高于人工栽培植株,约为栽培植株的1.8倍,其他2种物质含量以人工栽培略高.不同部位、冠层样品中3种活性物质含量差异显著,以茎皮、高冠层枝叶含量较高.不同年限南方红豆杉植株中紫杉醇、7-木糖紫杉醇和三尖杉宁碱含量以4年生植株较高,分别为0.08、0.91和0.32 mg·g-1.不同坡向植株中3种活性成分含量以阳坡植株较高,紫杉醇和7-木糖紫杉醇含量差异显著,三尖杉宁碱含量差异不显著.南方红豆杉中3种活性成分的积累与光照条件密切相关,栽培过程中适当增加光照有利于3种活性成分的积累.     

发酵牛粪对黄粉虫幼虫生长发育的影响

为寻找大型养殖场牛粪资源化与产业化利用途径,本文探讨了杂食性黄粉虫转化与利用牛粪的可行性.将牛粪和黄粉虫常规饲料(65％麦麸、30％玉米面、5％豆粕)按梯度比例混合后,用有益微生物菌群(EM)发酵,筛选出FD1、FD2(牛粪含量分别为60％、80％)两组发酵饲料作为处理,以常规饲料为对照(CK)进行黄粉虫幼虫饲养试验,研究不同发酵料对黄粉虫幼虫生长曲线、死亡率、化蛹率、抗氧化系统等的影响,评价黄粉虫幼虫对发酵牛粪饲料的适应性.结果表明:与CK相比,虽然FD1处理黄粉虫幼虫生长周期延长了20 d,死亡率有所上升,但黄粉虫幼虫单体取食总量增加49％ (P＜0.01),末龄幼虫单体质量增加28％(P＜0.01),粗脂肪含量提高26％(P＜0.05),不饱和与饱和脂肪酸含量的比值比CK高32％(P＜0.05),抗氧化酶活性显著增强,对发酵饲料表现出良好的适应性;而FD2处理各项指标均比CK差,表现出较低的适应性.表明在FD1处理条件下,可利用黄粉虫3龄幼虫对牛粪进行资源化利用,具有较好的应用前景.     

Bi‐parental vs. cooperative breeding in a passerine: fitness‐maximizing strategies of males in response to risk of extra‐pair paternity?

In socially monogamous species, males that risk cuckoldry more than others might gain inclusive fitness by yielding paternity to relatives. We tested this prediction in the Tibetan ground tit Pseudopodoces humilis , an unusual facultative cooperative breeder wherein most helpers (87% males) join a mated pair shortly before clutch completion. Extra-pair paternity among bi-parental broods occurred less often (26% of broods, 9% of young) compared with cooperative broods (68%, 25%). In the former, most extra-pair sires (88%) were pair breeders unrelated to the cuckolded males, whereas in the latter, sires (87%) were mainly helpers related to the dominant males. Brood productivity did not differ between the bi-parental and cooperative breeders, but helpers' partitioning over group paternity reduced the realized reproductive success of helped males. After taking inclusive fitness into account, however, there was no difference in success of dominant males between the two social systems. One possible explanation for the differences in the rates of cuckoldry in the two systems was body size, because pair-bond males in bi-parental situations were significantly larger than those in cooperative ones. We propose two alternative strategies for males to maximize fitness: breed as a pair if large to avoid cuckoldry from helpers, or breed cooperatively if small but compromise some paternity to relatives. Our results provide an unusual route to the incidence of cooperative groups, based on constraints imposed by low competitive ability of breeding males rather than some external ecological or demographic factors.     

4种山药多糖的体外发酵特性比较研究

采用体外发酵技术评价分段醇沉得到的4个山药多糖对宁乡猪盲肠食糜体外发酵特性和发酵产物的影响,探讨其用作动物功能性营养调控剂的可行性.乙醇终浓度(%)分别为30、45、60和75时沉淀得到4个山药多糖,用硫酸蒽酮法测得其总多糖含量(%)分别为45.85、56.24、90.91和13.87.选用正常饲喂的肥育宁乡猪,处死后无菌取盲肠食糜作为接种物,分别以4个山药多糖和葡萄糖为底物进行体外发酵.于不同发酵时间点记录产气量,48 h后检测发酵液pH值、NH3和SCFA含量.结果表明,75%醇沉多糖发酵动力学参数最优,并极显著优于葡萄糖组(P<0.01);45%、60%醇沉多糖和葡萄糖组pH值最低,60%、75%醇沉多糖和葡萄糖组的NH3含量最低;葡萄糖组的乙酸、丁酸和异丁酸含量均显著高于其它处理组(P<0.05),60%醇沉多糖组的丙酸含量稍高于、丁酸和异丁酸含量略低于其它3个多糖组.丁酸和异丁酸含量与pH值和NH3含量呈显著相关.提示60%和75%醇沉山药多糖是2种理想的可被宁乡猪盲肠微生物利用的碳源,并可改变微生物产生的SCFA含量与组成.     

双波长法测定二元混合体系中戊糖的含量

目的:针对单波长法在测定己糖和戊糖的混合体系中戊糖含量时出现较大的误差,提出了等吸收双波长法测定二元混合体系中戊糖的含量.方法:通过对己糖(葡萄糖)和戊糖(木糖)的不同含量(5-150μg/体系)与显色剂反应产物在不同波长(500,～700nm)条件下吸光曲线以及显色剂本身吸光曲线进行综合考虑,确定双波长,并对单双波长法测定同样样品的回收率进行了比较分析.结果:确定的测定波长为670nm,参比波长为580nm,双波长法建立的标准曲线方程为:Y=0.0056x-0.0123(y为两个波长下吸光度的差值,x为木糖的含量(μg)).R2=0.9995,线性范围为5-30μg/体系.单波长法对标样和试样的平均回收率分别为116%±15%和89%±13%;双波长法对标样和试样的平均回收率为104%±4%和95%±11%.结论:与单波长和一些其他方法相比,双波长法测定混合体系中戊糖含量较为简便易行且可以提高准确度.     

靶向ASGPR的反义核酸与抗HBV药物联合用药的体外抗HBV作用

研究以宿主基因ASGPR为靶的反义核酸与抗乙肝病毒(hepatitis B virus,HBV)药物联合应用的抗-HBV作用,为HBV感染的联合治疗和用药提供新的思路。以HepG2.2.15细胞为靶细胞,脂质体为载体将靶向ASGPR的硫代反义寡核苷酸(ASODN)转染至HepG2.2.15细胞中,6h后加入抗病毒药拉米夫定(3TC)或阿地福韦(ADV),72h后收集细胞培养液,采用酶联免疫法及荧光定量PCR法测定ASODN与抗-HBV药物联合应用后对细胞培养液中HBsAg、HBeAg及细胞分泌HBV DNA的抑制作用。采用金正均Q值法对数据进行分析。拉米夫定(3TC)和阿地福韦(ADV)分别与ASODN联用对HBsAg的抑制呈相加或协同作用。随着ADV浓度的增加,两药的协同作用下降。3TC与ASODN联用对HBeAg的抑制呈拮抗、相加和协同作用,其拮抗作用随着3TC浓度的增加逐渐减弱。ADV与ASODN联用对HBeAg的抑制只呈相加作用。3TC和ADV分别与ASODN联用对HBV DNA的抑制均表现为相加作用或协同作用,取ADV0.5μmol/L与取ASODN0.2μmol/L联用时对HBV DNA的抑制率可提高到72.6%。ASODN在一定条件下分别与3TC及ADV联用有相加或协同抗HBsAg、HBeAg及HBV DNA的表达,尤以抗HBsAg作用显著增强。     

北京四座楼自然保护区植物区系及植被分析

通过样地法对北京市四座楼自然保护区的植被进行了调查,在本底调查的基础上分析了四座楼自然保护区维管植物的区系成分和植被分布规律：该保护区有北京新记录种1个：麦李（Prunus glandulosa Thunb．）;国家三级保护植物3种,即：紫椴（Tilia amurensis Rupr．）、黄檗（Phellodendron amurense Rupr．）和刺五加[Acanthopanax senticosus（Rupr．et Maxim．）Harms];并分布有较大面积的天然侧柏[Platycladus orientalis（Linn．）Franco]林。该保护区共有维管植物约869种,分别属于127科431属,其中蕨类植物共有16科19属33种,裸子植物共有3科4属4种,被子植物共有108科408属832种,其中野生植物有772种,占北京所具有的野生植物种类的1／3以上。四座楼自然保护区野生种子植物共有15个分布区类型,其中北温带分布类型最多,表明本保护区的植物分布具有温带性质。     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.