小鼠心脏发育的形态学研究

目的建立小鼠心脏正常发育的时间表以及对应的形态学特征模式.方法小鼠胚胎ED8.5、ED9.5、ED10.5、ED11.5、ED12.5、ED14.5、ED16.5、ED18.5和P1(postnatal day)(出生后1 d的仔鼠)标本,进行整体或心脏部位不同轴向切片,HE染色,采用PCTV图像分析系统,对各时相小鼠心脏形态发育特征进行研究.结果 ⑴细胞结构发育的时空模式:① ED8.5时,生心板形成;ED9.5时,心肌细胞呈不规则的纺锤形,细胞的大小多样化,细胞核小;ED10.5时,小血管和着色较浅的肌原纤维出现,细胞之间连接较松;ED11.5时,心肌纤维排列较紧,纵断面上呈细长形,横断面上呈不规则多角形;ED12.5时,细胞核着色更清晰,心肌细胞形状逐渐规则,细胞之间紧密连接,同时闰盘结构出现在心室心肌细胞.②ED12.5时心肌小梁结构第一次在心室出现,ED14.5时增厚,而在心房少见心肌小梁.⑵心室结构的形成和心脏发育的成熟:①心肌间充质网络结构在ED10.5的心室中明显呈现,随着它的发育,心室的心内膜在ED11.5出现,心室心外膜可以辨认.②房室隔在ED12.5完全形成,心内膜垫在ED12.5开始发生并快速发育,促进室间隔在ED14.5完全形成.③心包膜在ED16.5可明显辨认,心包膜腔形成,此时近段流出道心内膜垫完全心肌细胞替换.结论肌原纤维细胞和心肌间充质细胞同时在ED10.5出现,提示肌原纤维对心肌细胞的成型和心肌化起作用.细胞的结构变化和心肌层的成熟过程,显示小鼠心脏部位成熟时间的不同,心室成熟相对较晚.     

鸡胚早期神经系统发育中凋亡细胞的分布研究

研究鸡胚18S（Stage）神经系统发育中凋亡细胞的分布及其生物学意义。采用HNK－1和TUNEL免疫组化双染及改变切片方向的方法观察了神经管和神经嵴的凋亡细胞,结果显示：凋亡的细胞在间隔10张横向切片上呈非均匀分布,但在矢状切面凋亡细胞有节段性分布趋势。体节处连续神经嵴没有发现凋亡细胞,而体节与体节之间神经嵴迁离神经管呈游离状并有凋亡细胞,神经管腹侧面体节处间充质细胞呈现细胞凋亡节段性分布。结果表明：鸡胚早期神经系统发育中选择性发生细胞凋亡作用。     

Cx43基因敲除鼠胚心脏近端流出道组织中Galpha13信号通路基因的差异表达

目的研究Cx43基因剔除(Cx43KO)小鼠胚胎心脏近端流出道组织中基因表达谱的改变,筛选可能导致Cx43KO小鼠流出道梗阻的相关基因。方法以胎龄(embryonic day,ED)14.5天的Cx43KO和野生型(Cx43WT)鼠胚心脏近端流出道部分为研究对象,分别提取总RNA,逆转录成cDNA;并在体外转录为cRNA,同时进行生物素标记及片段化;再与Affymetrix-4302.0基因芯片进行杂交。杂交信号经扫描后,应用相关生物信息软件分析基因表达情况。结果与Cx43WT组相比,Cx43KO组中表达上调2倍以上的基因共有287个,表达下调2倍以上的基因有199个。其中表达差异的基因参与转录调控、细胞周期等主要生理过程。进一步筛查表达差异1.5倍以上的基因发现,Galpha13信号通路上的多个基因在Cx43KO组有明显变化。结论利用基因芯片技术初步筛选出与Cx43KO鼠胚心脏近端流出道发育有关的多个基因,其中Galpha13信号通路上的相关基因可能与Cx43KO小鼠流出道梗阻的发生有关。     

心脏发育中的细胞凋亡

重点讨论了心脏发育过程中主要的细胞凋亡以及信号串级联,这些信号串往往伴随着有序的形态学上的事件.细胞凋亡主要发生在胚胎心脏中非心肌的隔室中,其隔室由心内膜、心外膜和神经嵴衍生的细胞组成.这些信号的级联包含了潜在生长转化因子的激活,导致心肌细胞的迁移以及随后的心内膜垫的心肌化.错误的细胞凋亡将导致心脏发育的异常.     

小鼠心脏发育中组蛋白乙酰化酶GCN5和PCAF的时空表达特征

目的研究组蛋白乙酰化酶氨合成通用控制蛋白5（general control nonderepressible-5 GCN5）和p300/CREB结合蛋白相关因子（p300/CREB binding protein-associated factor PCAF）在小鼠心脏发育过程中的时空表达规律。方法选取胎龄7.5-18d、出生1d和3月成年昆明小鼠正常心脏,利用免疫组织化学法和RT-PCR或Western blot技术半定量检测GCN5和PCAF在小鼠心脏发育过程中的时空表达变化。结果1.GCN5在E7.5-E9.5心脏原基中不表达;E10.5后心内膜垫及室间隔膜部和房室瓣相对强表达,心肌组织弱表达。GCN5 mRNA在E10.5-E11.5出现高峰表达,E12.5-E15.5表达水平下降,E16.5至成年鼠期表达极低。2.PCAF在E7.5-E9.5心脏原基中广泛弱表达;E10.5后心肌膜较强表达,心内膜、房室瓣和小梁网较弱表达,室间隔肌部弱表达,室间隔膜部极弱表达;PCAF蛋白在E10.5已有相对高表达,E11.5达到表达高峰,此后表达逐渐降低,在E13.5-E15.5和E16.5-生后1d仔鼠期出现两个表达平台期,成年鼠期心脏中表达最低。结论GCN5和PCAF在发育心脏中存在不同表达分布和变化规律,表明二者均参与了心脏的发生发育过程,且二者可能与心脏的特定结构发育密切相关。     

小鼠心脏神经嵴细胞的体外培养及其生物学特性

目的体外培养和鉴定心脏神经嵴细胞,探讨其生物学特性。方法取8·5d小鼠胚胎枕中部至第3体节神经管,组织块法无血清条件培养获心脏神经嵴细胞,采用转录激活因子2α(AP-2α)作为其生物学标记物,观察其迁移、分化等生物学特性。结果从胎鼠神经管中分离培养的细胞AP-2α表达阳性,具有迁移特性,传代后以含血清培养基培养后能自然分化成神经元和神经胶质细胞。结论体外培养可成功获得心脏神经嵴细胞,且具有迁移特性和多向潜能分化能力。     

心脏发育过程中的细胞命运转变及命运决定

胚胎发育过程中,心脏发生起源于生心中胚层(cardiac mesoderm)。在小鼠早期胚胎(E6.5),上胚体(epiblast)在低浓度Nodal诱导下, Eomes出现并激活Mesp1表达。Mesp1作为主调控者(master regulator),激活一系列生心关键转录因子及生心特异基因的表达,促进生心祖细胞的特化及生心区(cardiac field)的形成。之后的心脏形态发生涉及细胞命运的转变,包括流出道分隔过程中神经嵴细胞向间充质细胞转变、内皮细胞向间充质转变及房室通道发育过程中的内皮细胞向间充质转变。最新的研究表明,流出道在分隔成为主动脉和肺动脉根部之前,其中的细胞命运已经被预先设定。此综述文章重点探讨生心祖细胞特化、细胞命运转变与命运预先设定等方面的新进展,调控机制及争议问题。     

脂多糖LPS诱导的神经炎症对小鼠认知功能的影响及相关机制分析

为研究脂多糖(lipopolysaccharide,LPS)对小鼠认知功能的影响,本研究将18只C57BL/6小鼠随机分为两组,即正常对照组和LPS处理组,每组各9只小鼠。在腹腔注射LPS 24 h后行水迷宫实验检测小鼠行为学变化。此外,本研究采用Western blotting和免疫组化检测小鼠脑内小胶质细胞特异性标记物离子通道相关钙衔接蛋白(ionized calcium binding adapter,IBA1)的表达,TUNEL凋亡法检测小鼠脑内神经元凋亡情况。实验结果发现LPS处理后小鼠认知功能下降。Western blotting和免疫组化结果均提示LPS处理后小鼠脑中IBA1表达量增加,小胶质细胞激活;TUNEL提示LPS处理后小鼠脑内出现大量凋亡神经元,由此推断LPS可能通过激活小胶质细胞,扩大神经炎症反应,增加神经元凋亡导致小鼠认知功能下降。     

腺苷受体激动剂对心肌缺血再灌注损伤大鼠内质网应激的影响及其作用机制研究

目的:探讨腺苷受体激动剂对心肌缺血再灌注损伤(MIRI)大鼠内质网应激(ERS)的影响及其作用机制。方法:选取雄性成年Wistar大鼠56只,利用Langendorff装置制成大鼠离体心脏MIRI模型。随机分为四组(n=14):假手术组(Sham组)、心肌缺血再灌注组(MIRI组)、腺苷受体激动剂组(NECA组)和内质网应激抑制剂组(TUDCA组)。利用透射电镜观察四组心肌超微结构的变化;免疫组化观察心肌肌醇依赖酶1α(IRE1α)的表达情况;Western blot方法检测心肌ERS中IRE1-XBP1信号通路标志蛋白IRE1α、X盒结合蛋白1s(XBP1s)的表达水平;TUNEL检测心肌细胞凋亡情况。结果:透射电镜结果显示,Sham组肌丝排列规则致密,嵴排列整齐,外膜和肌节形态完整;MIRI组大部分肌丝断裂,肌节挛缩变形,嵴排列稀疏结构破坏,间隙增宽,可见线粒体空泡变性;NECA组及TUDCA组较MIRI组损伤减轻,内质网轻度扩张或者正常,肌丝排列较整齐。免疫组化结果发现,Sham组心肌纤维呈细长圆柱形,形态正常,少量结缔组织存在,基本无IRE1α阳性染色;MIRI组细胞排列紊乱,有许多断裂的细胞出现,IRE1α阳性染色区域显著增加,而NECA和TUDCA组细胞病理的变化较轻,相对于MIRI组,IRE1α阳性染色部位明显减少。Western blot结果显示,与Sham组相比,MIRI组的IRE1α和XBP1s蛋白的表达水平明显上升(P0.05);而与MIRI组相比,TUDCA组及NECA组IRE1α和XBP1s的蛋白表达水平显著降低(P0.05)。TUNEL结果显示,MIRI组细胞凋亡明显,Sham组基本没有发生心肌细胞凋亡,MIRI组较NECA组及TUDCA组的凋亡细胞数更多。结论:NECA可通过抑制IRE1-XBP1信号通路来减轻ERS反应,达到保护心肌组织细胞的目的。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.