原子力显微镜在植物学研究中的应用

原子力显微镜(AFM)是一种探测样品表面信息的有力工具, 它可以在空气和接近样品生理条件下成像, 同时也可以在皮牛(pico-Newton, 10-12 N)至微牛(micro-Newton, 10-6 N)水平上测量力的大小。本文主要介绍了自AFM发明以来, 其在植物大分子、细胞器、细胞、叶片等方面的应用, 并列举了目前 AFM存在的几点不足。     

用原子力显微技术研究受体-配体间的相互作用

原子力显微镜(AFM)不仅能对纳米生物结构进行实时动态的形态和结构观察,而且还能以10^-12N(pN)的精度对溶液中生物分子表面的相互作用力进行直接测量,逐渐成为一种研究受体-配体间相互作用的良好工具。本简要综述用AFM研究受体-配体间作用力、受体-配体间相互作用的影响因素及对这些因素的处理方法。     

茉莉酸甲酯对水稻幼苗抗冷性的影响

测定茉莉酸甲酯（MJ）对冷胁迫条件下水稻幼苗生长和与抗冷性相关生理生化指标影响的结果表明：较高浓度的MJ（10^-3mol·L^-1）明显抑制冷胁迫下的水稻幼苗生长,增加细胞膜的渗透性;浓度低一些的MJ（10^-4、10^-5、10^-6、10^-7mol·L^-1）对冷胁迫下水稻幼苗的生长和细胞膜有不同程度的保护作用,其中以10～mol·L^-1MJ的效果为佳。10～mol·L^-1 MJ处理的叶中叶绿素降解程度小,可溶性糖、可溶性总蛋白和脯氨酸的含量以及超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性均增加,丙二醛（MDA）含量降低,因而水稻幼苗的抗冷性提高。     

琥珀酸对大鼠海马CA1区神经元电压依赖性钙电流的影响

目的：观察不同浓度的琥珀酸对大鼠海马CA1区神经元电压依赖性钙通道（voltage—dependent calcium channels,VDCC）电流的作用,初步探讨琥珀酸对神经元保护的电生理学基础。方法：采用传统全细胞膜片钳技术和制霉菌素（nystatin）穿孔膜片钳技术观察琥珀酸对海马CA1区神经元VDCC电流的影响。结果：不同浓度的琥珀酸（10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1）在海马CA1区对低电压激活（low—voltage activated,LVA）钙通道电流未见任何影响,而对高电压激活（high—voltage activated,HVA）钙通道电流的抑制呈浓度依赖性。对照组HVA钙电流为580．05±17．32pA,分别给予10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1。的琥珀酸后,HVA钙电流依次为563．74±16．65,517．99±15．24,444．66±13．26,405．32±19．11,269．03±9．96和86．41±3．25pA,同对照组相比差异有统计学意义（n=8,P〈0．01）。结论：琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响。由此推测琥珀酸可能通过抑制HVA钙电流减少Ca^2＋内流而影响海马CA1区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关。     

一氧化氮在17-β雌二醇抑制血管平滑肌细胞增殖和原癌基因c -fos表达中的作用

实验利用大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC)作为模型,观察17-β雌二醇（E2）对VSMC增殖和原癌基因c-fos表达的影响,并探讨VSMC源性一氧化氮（NO）在基中的作用,检测指标包括NO释放的测定,细胞计数、^3H-Tdr掺入,噻唑蓝（MTT）测定和c-fosmRNA表达,结果显示,E2（10^-12-10^-8mol/L)呈浓度依赖性地促进VSMC中NO的释放;10^-8mol/LE2能明显抑制10%小牛血清（FCS）和10^-7mol/L内皮素-1（ET-1）诱导的细胞增殖和DNA合成,E2的抑制作用均可被雌激素受体（ER）拮抗剂tamoxifen(10^-7mol/L)和一氧化氮合酶抑制剂L-NAME（10^-6mol/L)明显减轻;E2（10^-8mol/L)可明显抑制10^-7mol/LET-1诱导的VSMCc-fos表达,这种抑制作用可被L-NAME（10^-6mol/L)明显减轻,这些结果提示E2能抑制VSMC增殖和原癌基因c-fos表达,这种促进VSMC的NO释放密切相关,而且该作用至少部分通过ER介导。     

检测PCV2、PPV、PRV疫苗株与野毒株的多重PCR方法

本文建立了一种同时检测猪圆环病毒2型（PCV2）、细小病毒（PPV）、及伪狂犬病毒（PRV）疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp（PCV2）、581bp（PPV）、372bP（PRV gB）及147bp（PRV gE）四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10^-6.2、10^-3.8、10^-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.     

PGE2、PGF2α和Iloprost对小鼠2-细胞胚胎体外发育的影响

目的研究PGE2、PGF2。以及Hoprost（PGl2的稳定类似物）对小鼠2-细胞胚胎体外发育的影响。方法在含0．5％BSA的mCZB液中分别添加0、10^-4、10^-5和10^-6mol／L的PGE2,0、10^-4、10^-5和10^-6mol／L PGF2α以及0、1×10^-6、2×10^-6和4×10^-6mol/L Iloprost,观察小鼠2-细胞胚胎的体外发育,同时利用H33342染色进行囊胚和孵化囊胚的细胞核计数。结果添加PGE2、PGF2α以及Iloprost的各处理组2-细胞胚胎体外培养的囊胚率和孵化率均显著低于对照组（P〈0．05）,但各处理组与对照组之间在囊胚或孵化胚胎的细胞数上差异不显著（P〉0．05）。结论培养液中添加这三种前列腺素均不利于小鼠2-细胞胚胎的体外发育,但不影响囊胚或孵化胚胎的细胞数。     

15-KETE对慢性缺氧大鼠肺动脉平滑肌细胞钾离子通道的作用

目的观察15-酮基二十碳四烯酸（15-ketoeicosatetraenoic acid,15-KETE）对缺氧大鼠肺动脉平滑肌细胞（pulmonary arterial smooth cells,PASMCs）膜电压门控钾离子通道的活性的影响。方法将12只雄性SD大鼠随机分成对照组和缺氧组,每组6只。采用急性酶分离法（胶原酶Ⅰ型和弹性酶）获得SD大鼠单个PASMCs,应用全细胞膜片钳记录方法,研究15-KETE对两组大鼠膜电位（Em）、膜电容（Cm）、电压门控钾电流（IKv）的影响。结果 （1）慢性缺氧使大鼠PASMCs的Em显著去极化（P〈0.05,n=6）,明显地抑制了大鼠PASMCs的IKv（P〈0.01,n=6）,对大鼠PASMCs的Cm无影响;（2）较高浓度的15-KETE（1×10^-7 mol/L、1×10^-6 mol/L）可使慢性缺氧大鼠PASMCs去极化;（3）15-KETE（1×10^-8 mol/L～1×10^-6 mol/L）可浓度依赖性地抑制慢性缺氧大鼠PASMCs的IKv;（4）较高浓度15-KETE（1×10^-7 mol/L、1×10^-6 mol/L）对缺氧PASMCs IKV的平均阻抑率显著高于常氧PASMCs。结论缺氧未改变15-KETE引大鼠PASMCs去极化及浓度依赖抑止IKv的特性,且缺氧可能改变了PASMCs对15-KETE的敏感性。     

不同形态氮素培养下水稻叶片中蛋白质差异表达

硝态氮除作为主要氮源外,还作为一种潜在的信号物质,在植物生长发育过程中起着重要作用,其作用方式的直接性或间接性近年来成为了研究热点。利用双向电泳（2-DE）技术,对不同形态氮素（NO3^-与NH4^＋）培养的水稻叶片蛋白表达谱进行了比较分析,结果在硝态氮和铵态氮培养的叶片中分别分辨出26个和6个增量表达蛋白。质谱分析结合数据库检索鉴定出11个蛋白,其中7个硝态氮上调的蛋白为：光系统Ⅱ放氧复合蛋白1（N1）、抗性相关蛋白MLA13（N2）、光系统Ⅱ23kD多肽（N3）、翻译激活因子（N5）、光系统Ⅱ放氧复合蛋白2前体（N8）、未知蛋白（N17）和泛素载体蛋白（N18）;4个铵态氮上调蛋白为：ATP合酶β亚基（A1）、转氨酶（A3）和两个功能未知的蛋白（A5,A6）。上述研究结果有助于了解水稻适应不同氮素营养时的生物化学基础及可能的生物学意义,同时也为深入阐明水稻响应NO3^-与NH4^＋信号的反应提供了蛋白水平的证据。     

血管钠肽抑制低氧刺激心脏成纤维细胞增殖的机制研究

目的：研究血管钠肽（VNP）抑制低氧刺激的心脏成纤维细胞增殖的机制。方法：发离、培养乳鼠心脏成纤维细胞,随机分为四组：对照组、低氧组、低氧＋VNP组和低氧＋8－Bromo-cGMP组。以MTT法观察各组细胞的生长情况,分别采用放射免疫和免疫组化的方法研究了VNP对细胞内cGMP水平和增殖细胞核抗原（PCNA）表达的影响。结果：低氧24h可以使培养的乳鼠心脏成纤维细胞MTT A490nm值显著升高（P＜0．05vs对照组）,VNP（10^-7mol/L和8－Bromo-cGMP(10^-3mol/L)均可以显著降低低氧刺激的心脏成纤维细胞MTT A490nm值（P＜0．05vs低氧组）;对照组和低氧组细胞内cGMP水平无显著差异,而VNP（10^-7mol/L）能升高细胞内cGMP水平（P＜0．05vs对照组、低氧组）;低氧组PCNA的表达显著强于对照组（P＜0．05vs对照组）,VNP（10^-7mol/L可以使低氧刺激的心脏成纤维细胞PCNA表达减弱（P＜0．05vs低氧组）。结论：VNP抑制低氧刺激的心脏成纤维细胞增殖与升高细胞内cGMP水平、减弱PCNA的表达有关。     

Imaging the ordered arrays of water-soluble protein ferritin with the atomic force microscope

Individual water-soluble molecules of the protein ferritin have been imaged on a silicon surface in pure water at room temperature with the atomic force microscope (AFM). The ferritin molecules formed an ordered monolayer by binding to a charged polypeptide monolayer of poly-1-benzyl-L-histidine (PBLH) spread at the air-water interface. The film, fully wetted with water, was horizontally transferred onto an alkylated silicon wafer for AFM imagings. The hexagonal arrangement of ferritin molecules was imaged with high reproducibility on the whole surface of the film, since the forces between cantilever and the sample could be kept sufficiently smaller than 10^-10 N, mainly due to a “self-screening effect” of the surface charges of the ferritin-PBLH layer. This is the first observation of two-dimensional ordered arrays of water-soluble protein molecules directly confirmed by AFM with molecular resolution.     

Lateral mechanical coupling of stereocilia in cochlear hair bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).     

Assessment of elasticity and topography of Aspergillus nidulans spores via atomic force microscopy

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA(+) and rodA(-) strains. Tapping-mode AFM images of wild-type and rodA(+) spores in air showed characteristic "rodlet" protein structures covering a granular spore surface. In comparison, rodA(-) spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA(+) spores. Rodlets were removed from rodA(+) spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 +/- 10, 120 +/- 10, and 300 +/- 20 N/m and the elastic moduli to be 6.6 +/- 0.4, 7.0 +/- 0.7, and 22 +/- 2 GPa for wild-type, rodA(+) and rodA(-) spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

Disposable biosensor based on graphene oxide conjugated with tyrosinase assembled gold nanoparticles

A highly efficient enzyme-based screen printed electrode (SPE) was obtained by using covalent attachment between 1-pyrenebutanoic acid, succinimidyl ester (PASE) adsorbing on the graphene oxide (GO) sheets and amines of tyrosinase-protected gold nanoparticles (Tyr-Au). Herein, the bi-functional molecule PASE was assembled onto GO sheets. Subsequently, the Tyr-Au was immobilized on the PASE-GO sheets forming a biocompatible nanocomposite, which was further coated onto the working electrode surface of the SPE. The characterization of obtained nanocomposite and modified SPE surface was investigated by atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Attributing to the synergistic effect of GO-Au integration and the good biocompatibility of the hybrid-material, the fabricated disposable biosensor (Tyr-Au/PASE-GO/SPE) exhibited a rapid amperometric response (less than 6s) with a high sensitivity and good storage stability for monitoring catechol. This method shows a good linearity in the range from 8.3×10(-8) to 2.3×10(-5) M for catechol with a squared correlation coefficient of 0.9980, a quantitation limit of 8.2×10(-8) M (S/N=10) and a detection limit of 2.4×10(-8) M (S/N=3). The Michaelis-Menten constant was measured to be 0.027 mM. This disposable tyrosinase biosensor could offer a great potential for rapid, cost-effective and on-field analysis of phenolic compounds.     

Unfolding and extraction of a transmembrane alpha-helical peptide: dynamic force spectroscopy and molecular dynamics simulations

An atomic force microscope (AFM) was used to visualize CWALP(19)23 peptides ((+)H(3)N-ACAGAWWLALALALALALALWWA-COO(-)) inserted in gel-phase DPPC and DSPC bilayers. The peptides assemble in stable linear structures and domains. A model for the organization of the peptides is given from AFM images and a 20 ns molecular dynamics (MD) simulation. Gold-coated AFM cantilevers were used to extract single peptides from the bilayer through covalent bonding to the cystein residue. Experimental and simulated force curves show two distinct force maxima. In the simulations these two maxima correspond to the extraction of the two pairs of tryptophan residues from the membrane. Unfolding of the peptide precedes extraction of the second distal set of tryptophans. To probe the energies involved, AFM force curves were obtained from 10 to 10(4) nm/s and MD force curves were simulated with 10(8)-10(11) nm/s pulling velocities (V). The velocity relationship with the force, F, was fitted to two fluctuation adhesive potential models. The first assumes the pulling produces a constant bias in the potential and predicts an F approximately ln (V) relationship. The second takes into account the ramped bias that the linker feels as it is being driven out of the adhesion complex and scales as F approximately (ln V)2/3.     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.