银合欢根瘤细胞的光学显微镜和电子显微镜研究

银合欢接种根瘤菌形成根瘤后,应用光镜和电镜技术观察。银合欢根瘤由分生组织细胞、皮层组织细胞、维管束系统和侵染细胞区域四个不同部分组成。根瘤菌借助于侵染线侵染细胞,释放进入宿主细胞质中,转变成固氮类菌体。最初每个包被膜内只含单独的类菌体,随后较老的侵染细胞中,每个包被膜内含有一个以上的类菌体。因此,成熟根瘤的侵染细胞可见有2~5个类菌体群集包被膜里,并且明显地累积PHB物质,显示电子染色透明颗粒。本文还讨论了上述变化的意义与银合欢根瘤细胞结构和功能的关系。     

肯氏相思根瘤亚显微结构观察

肯氏相思根瘤亚显微结构观察结果表明,刚受侵袭的寄主细胞的细胞核和质体膨大,线粒体内嵴消失成为圆球状体,内质网膜松散;幼年类菌体细胞外形较小,呈圆形和椭圆形,细胞质浓密,染色深而均匀;成熟类菌体外型较大,形态多样,细胞内聚－β－羟基丁酸积累增多,随着根瘤细胞逐渐发育成熟,在类菌体被内可见１至数个类菌体,在根瘤衰老细胞中,类菌体包被周膜解离破裂,流出电子透明物质,本文还对周膜扩增的问题进行了讨论。     

超结瘤大豆根瘤的亚显微结构

电镜观察表明,超结瘤大豆未受侵染的宿主细胞中有一明显增大的细胞核。幼年类菌体为椭圆形,里面有个拟核区,正常类菌体有完善的周膜和ＰＨＢ颗粒,受侵染的寄主细胞中出现类似无效根瘤的异常现象;少数类菌体退化或溶解,还有空周膜及裸露的类菌体,这可能是超结瘤大豆固氮活性较低的原因。     

超结瘤大豆根瘤的亚显微结构

电镜观察表明,超结瘤大豆未受侵染的宿主细胞中有一明显增大的细胞核。幼年美菌体为椭圆形,里面有个拟核区,正常类菌体有完整的周膜和PHB颗粒。受侵染的寄主细胞中出现类似无效根瘤的异常现象：少数类菌体退化或溶解,还有空周膜及裸露的类菌体,这可能是超结瘤大豆固氮活性较低的原因。     

灰金合欢根瘤类菌体繁殖及其宿主细胞的超微结构

根瘤细胞早期发育阶段,以宿主细胞器和根瘤菌转化类菌体的数量增多为特征。随后类菌体增殖到填满宿主细胞内的大部分区域。各个类菌体周膜内含有1至几个类菌体。晚期共生发育阶段,类菌体细胞结构和宿主细胞器数量发生了变化。文中还讨论了根瘤的共生固氮作用。     

肯氏相思根瘤亚显微结构观察

肯氏相思根瘤亚显微结构观察结果表明：刚受侵染的寄主细胞的细胞核和质体膨大,线粒体内嵴消失成为圆球状体,内质网膜松散;幼年类菌体细胞外型较小,呈圆形和椭圆形,细胞质浓密,染色深而均匀;成熟类菌体外型较大,形态多样,细胞内聚-β-羟基丁酸（PHB）累积增多;随着根瘤细胞逐渐发育成熟,在类菌体包被内可允1至数个类菌体;在根瘤衰老细胞中,类菌体包被周膜解离破裂,流出电子透明物质。本文还对周膜扩增的问题进行了讨论。     

2,4-D诱发小麦根瘤及引导根瘤菌进入小麦根瘤细胞的研究

本文通过水培和砂培的冬小麦(丰抗8号),研究用2,4-D处理并接种慢生型大豆根瘤菌61A76(Bradyrhizobium japonicum 61A76)和快生型沙打旺根瘤菌16(Rhitobium sp. astrugllus16),诱发小麦形成含菌根瘤的可能性。结果表明2,4-D可诱发小麦根系产生瘤状结构,此瘤状结构起始于根中柱鞘,2,4-D刺激小麦根瘤细胞的DNA合成,根瘤细胞的核及DNA含量约为正常根部的二倍,2,4-D能引导上述两种根瘤菌进入小麦根瘤细胞并大量繁殖,进入根瘤细咆的根瘤菌其形态有些变化,内含聚β—羟丁酸颗粒,有些菌体被膜囊包围。从接种大豆根瘤菌的小麦根瘤内分离出一株根瘤菌,经镜检、血清学及回接大豆试验证明为原接种的大豆根瘤菌株。     

沙棘根瘤内生菌的多型性

用透射电子显微镜观察了春、夏、秋、冬四个季节的沙棘根瘤,以及瘤瓣上、中、下三个部位。结果表明,不同季节,不同部位的瘤瓣内,根瘤内生菌有7种不同形态。即侵染菌丝体、繁殖菌丝体、营养菌丝体、春孢子及春孢子囊、泡囊,冬孢子及冬孢子囊和类菌体。在多年生珊瑚状的根瘤中,它们的世代交替是:春夏季以侵染菌丝、繁殖菌丝、营养菌丝、春孢子囊及春孢子、泡囊为主;秋冬季以衰退的营养菌丝、衰老泡囊、冬孢子囊和冬孢子、类菌体为主。冬孢子和类菌体是休眠体。     

豆科根瘤内气体交换和气体扩散模型研究

近年来的研究表明根瘤皮层内存在着可调节的气体扩散屏障,它是由根瘤皮层内的一层细胞及填充在胞间隙的水层构成的,而根瘤是通过改变填充该层胞间隙的水层厚度来调节对气体扩散的阻力。本文概述了关于模拟豆科根瘤内气体交换和气体扩散的数学模型研究,阐明调节根瘤内含类菌体细胞维持低氧分压的有关问题。模型研究使我们获得了对共生固氮根瘤内极为复杂的微生态环境的初步认识,有待于通过改进试验和借助其他理论进一步探索根瘤气体交换和气体扩散的本质。     

与玉米混作改善花生铁营养对其根瘤形态结构及豆血红蛋白含量的影响

通过土培方法研究了与玉米混作对花生根瘤形态结构及固氮功能的影响。结果表明,玉米与花生混作能够明显地改善花生铁营养、提高根瘤豆血红蛋白的含量。同时,单作花生根瘤细胞液泡化程度较高,正在发育的根瘤细胞内类菌体数量明显地比混作的花生低。成熟根瘤细胞类菌体周膜外空间(细胞壁以内、周膜外的空间)体积变大。说明单作花生固氮酶活性较低的原因是缺铁抑制了豆血红蛋白的合成和改变了根瘤形态结构以及类菌体的超微结构。     

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution¹. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe''s lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature². The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 μm is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 μm)³. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Spin label study of erythrocyte deformability I. Electron spin resonance spectral change under shear flow

A spin labeling method in electron spin resonance spectroscopy (ESR) is applied for the first time to study the deformability of human red blood cells (RBC). ESR measurements of a RBC suspension incubated with a fatty acid spin label were performed, using a narrow-gap flat ESR sample cell under various flow shear stresses (tau). Remarkable changes were observed in ESR spectra with tau, indicating that RBC are oriented in such a way that the greater part of the membrane surface is aligned parallel to the ESR cell walls. The diamide-treated, hardened RBC, in which the biconcave discoid shape remains intact under no shear stress, exhibit a smaller ESR spectral change with tau than the intact, demonstrating that the present method can be used to assess the deformation of RBC occurring with flow orientation. In particular, the relative amplitude of an ESR difference spectrum may be used as a measure of the elongation of RBC. The conclusion is further supported by experiments using glutaraldehyde-treated or heat-denatured RBC. All these ESR results are in good agreement with the corresponding results obtained by several different methods. The present spin labeling technique is thus proven to be applicable for evaluating RBC deformability.     

Use of a Low-Frequency Esr Spectrometer: Implications for Spin-Trapping Free Radicals, In Situ

We have adapted the low-frequency ESR spectrometer, designed and built by H.J. Halpern, to the physiologic needs of organ preparations operating at 250 MHz. Initial studies have allowed us to detect nitroxides in an isolated perfused heart. These in siru measurements were made with nitroxides specifically designed to mimic the lipophilic nature of 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) and 2.2-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH). These spin labels provided information about the influence of dynamic factors of the heart, such as flow rate, different cell populations and unequal distribution between compartments on our ability to conduct and interpret spin trapping experiments. They also clarified the sacrifice in sensitivity involved in operating at the lower frequencies. To deal with this later problem. we have increased the sensitivity of the spin trapping method by synthesizing a family of ¹⁵N-and deuterium-containing DMPO analogs and by determining their ability to spin trap free radicals generated by the model superoxide system of xanthinelxanthinc oxidase. Finally, since activated neutrophils are one of the few cells known to generate free radicals as part of their physiologic function, we used these phagocytic cells, as a source of superoxide.     

DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra

Non-viral vectors represent an important alternative in gene delivery. Among these vectors, cationic liposomes are widely studied, because of their ability to form stable complexes with DNA fragments (lipoplexes). In the present work, we report on the characterization by electron spin resonance (ESR) spectroscopy and zeta potential measurements of cationic liposomes and of their complexes with oligonucleotides. Liposomes were made with a zwitterionic lipid, DOPE, and a cationic lipid, either DOTAP or DC-Chol. Oligonucleotides were the 20-base single strand polyA, the 20-base single strand polyT, and the corresponding double strand dsAT. The zeta potential as a function of the oligonucleotide/lipid⁺ ratio gave an S-shaped titration curve. Well-defined surface potential changes took place upon charge compensation between the cationic lipid heads and the phosphate groups on the oligonucleotides. The inversion point depended on the specific system under study. The bilayer properties and the changes that occurred with the incorporation of DNA fragments were also monitored by ESR spectroscopy of appropriately tailored spin probes. For all the systems investigated, the ESR spectra showed that no major alteration took place after lipoplex formation and molecular packing remained substantially unchanged. Both zeta potential and ESR measurements were in favor of an external mode of packing of the lipoplexes.     

The interaction of the cell-penetrating peptide penetratin with heparin, heparansulfates and phospholipid vesicles investigated by ESR spectroscopy.

An ESR investigation of the interaction of spin-labelled penetratin with heparin, heparansulfates and several phospholipid vesicle formulations is reported. Penetratin is a 16-aa peptide corresponding to the third helix of the Antennapedia homeodomain and belonging to the cell-penetrating peptide family. The present study shows that ESR spectroscopy can provide specific and reliable information about the mechanism of interaction of penetratin with polysaccharides and lipids, at a molecular level. The study showed that: (i) heparin and heparansulfates specifically interact with spin-labelled penetratin and promote peptide aggregation and concentration on their molecular surface; (ii) penetratin does not interact with neutral lipids, whereas it enters negatively charged lipid bilayers; (iii) cholesterol plays a negative effect on the insertion of penetratin into the lipid membrane; (iv) the interaction of penetratin with lipid vesicles is strongly dependent on lipid concentration. In a low lipid regime, penetratin associates with the polar heads of phospholipids and aggregates on the membrane surface; once the lipid concentration attains a threshold, the peptide enters the lipid bilayer. This step is characterized by reduced peptide mobility and partial disaggregation.It has been shown that ESR spectroscopy is a valuable investigation tool in studies related to the still unclear mechanism of the internalization process.     

ESR Spectroscopy Investigation of the Denaturation Process of Soybean Peroxidase Induced by Guanidine Hydrochloride, DMSO or Heat

The involvement of protein denaturation and/or misfolding processes in the insurgence of several diseases raises the interest in structural dynamic studies of proteins. The use of nitroxide spin labels with electron paramagnetic resonance is a powerful tool for detecting structural changes in proteins. In the present study, we apply this strategy to soybean peroxidase (SBP), a protein characterised by high thermal and structural stability, and we propose a simple method to analyse the anisotropy changes of the protein system and to relate them with the structural changes induced by protein unfolding. We examined the effect of temperature, guanidine hydrochloride and dimethylsulfoxide on the stability of SBP and looked for correlations between the ESR results and the experimental findings obtained by other techniques, reported in the literature. The agreement between data obtained through different strategies supports the validity and reliability of the ESR approach to protein unfolding.     

ESR technique for noninvasive way to quantify cyclodextrins effect on cell membranes

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe, which can be further correlated with the microviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes.     

Fluorescence and ESR spectroscopy studies on the interaction of isoflavone genistein with biological and model membranes

Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein.     

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.