Application of cryosubstitution in neurohormone-and neurotransmitter-immunocytochemistry

Summary Pituitaries of the African catfish, Clarias gariepinus, were prefixed in aldehyde fixatives, frozen in liquid propane and submitted to a cryosubstitution procedure. Ultrathin sections of the Lowicryl HM20-embedded tissue were treated with primary antisera raised in rabbits to gonadotropin releasing hormone (GnRH), vasopressin or gamma amino butyric acid (GABA) respectively. Binding of the primary antisera was visualized with goat anti-rabbit (GAR) labeled with gold. The general morphology of the tissue components in the cryosubstituted pituitaries matches with that obtained after routine embedding procedures. In addition, a strong labeling intensity of the neuropeptides/neurotransmitters investigated in the present study was demonstrated. Due to these qualities cryosubstitution provides optimal conditions for studying co-localization of neurosecretory products, using double-immunostaining procedures. In the pars distalis of the catfish pituitary several types of hypothalamus-derived nerve fibers are present between or synapting on the secretory cells. It is demonstrated that the two known catfish GnRHs are co-localized in the same nerve fiber and within these nerve fibers even co-exist in the same neurosecretory granules. GABA and vasopressin-immunolabeling each occurred in different nerve fibers. The present data demonstrate that cryosubstitution and low temperature-embedding results in an excellent morphological preservation compared to ultracryotomy and a better preserved immunoreactivity of small antigenic molecules in comparison to conventional fixation and embedding techniques.     

Some applications of cryosubstitution in ultrastructural studies of the cell nucleus.

Cryofixation followed by cryosubstitution, without the use of any chemical fixatives, was carried out on cultured mouse P815 cells. The principal aim of our work, which was to show that these techniques provide excellent morphological preservation of cellular and in particular nuclear components, was demonstrated. All nuclear structural components, nucleolar or nucleoplasmic, were clearly revealed using this technology. The cells were cryofixed by impact freezing onto a copper mirror cooled with liquid nitrogen or helium, cryosubstituted in acetone and embedded in either Lowicryl K11M or 1R White acrylic resin. Ultrathin sections were contrasted using either the usual uranyl acetate-lead citrate double staining, a differential staining for nuclear nucleoprotein structures, or the silver staining revealing nucleolar organizer regions. In view of the absence of conventional fixatives, the specimens prepared in this way would offer to be material of choice for ultrastructural identification of intra-nuclear antigens, especially those sensitive to conventional fixatives such as, for example, aldehydes. Advantages and differences of these techniques with regard to more conventional electron microscopic procedures are discussed.     

Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution.

Escherichia coli cells were very rapidly frozen and substituted at a low temperature with 3% glutaraldehyde in acetone. Infiltration and embedding with Lowicryl K4M were carried out at -35 degrees C. This procedure resulted in good structural preservation of both the nucleoid morphology and its DNA plasm, such that immunolabeling with the protein-A gold technique could be carried out. With antibodies specific for either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), it was shown that dsDNA was present throughout the nucleoid but that ssDNA was located on the nucleoid periphery. Chloramphenicol-treated cells, in which protein synthesis but not DNA replication is stopped, produced a characteristic ringlike nucleoid shape and had both dsDNA and ssDNA present throughout the annular section of the DNA plasm. The relationship between metabolically active DNA and overall bacterial genome organization is discussed.     

Methods of quick-freezing and cryosubstitution of biological tissues in analytical ion microscopy

The localization and relative concentration of diffusible ions (Li+, K+) or bound but easily displaceable ions (Ca2+) have been studied in myocardium prepared by rapid-freeze fixation and compared with results from chemically prepared samples. Contrary to chemical fixation which gives diffused potassium imaging, cryofixation enables elemental precise mapping in cellular compartment. Bound displazable external calcium, extracted by chemical fixation, is visualized after rapid-freeze fixation. Its localization is distinguished by a bright cellular outline, which corresponds to the glycocalix. Intracellular calcium is less emissive and has the same distribution and concentration whatever the sample preparation. Very movable and present at low concentration in tissues, lithium is localized after rapid-freeze fixation. Those qualitative and quantitative results show the adequation of rapid freeze fixation and freeze substitution to the study of diffusible ions by means of analytical ion microscopy.     

Ultrastructural analysis ofErysiphe graminis haustoria and subcuticular stroma ofVenturia inaequalis using cryosubstitution

Summary Cryosubstitution provides an improved ultrastructural preservation of the two plant pathogensVenturia inaequalis andErysiphe graminis when compared to conventional preparation methods. Further, freezing the infected whole leaf material on a copper block cooled with liquid helium gave better results than those observed with the propane plunging method. Novel observations concerning the fungal stroma and haustoria were made which showed ribosomes organized into groups that were evenly distributed throughout the cytoplasm of both fungi. Stretches of rough endoplasmic reticulum were present, and microtubules were seen, sometimes associated with mitochondria. A large number of darkly staining vacuoles were observed in both fungi. The polarity of organelles and microtubules along the longitudinal axis of the haustorial body ofE. gramnis and along the growing direction of subcuticular stroma and runner hypha ofV. inaequalis was evident. InE. graminis filasomes were observed, as were Golgi-like bodies. These new observations, together with the advantages of the cryosubstitution technique, can serve as a basis for further studies in understanding host-parasite interactions.     

(3-Cryo) methods (cryofixation, cryosubstitution and cryoembedding) for processing of tissues for ultrastructural and immunocytochemical studies. Application to oviduct cells of laying quail.

Cryomethods occupy a privileged position among the procedures used for the preparation of biological samples for the various studies that may be performed in electron microscopy (ultrastructural, immunocytochemical and microanalysis in situ). In general, cryomethods are specific to one, or a maximum of 2 types of application. The (3-Cryo) methods (cryofixation, cryosubstitution without fixatives and cryoembedding in the new Lowicryl resins (K11M or HM23) are a set of methods for correlating new structural information with analytical and biochemical data. However, these 3-Cryomethods are delicate, complicated and expensive. To demonstrate that they can be performed, at least in part, with home-made systems at a reasonable cost, we have carried out a structural and immunocytochemical study on the oviduct of the laying quail. We studied the localization of 2 proteins, one cytoplasmic (ovalbumin) and the other nucleolar (B-36). The results provided by the 3-Cryomethods are compared with those obtained with other immunocytochemical methods, including tissue processed by conventional chemical fixation and high or low temperature embedding, or by 2-Cryomethods (cryofixation and cryosubstitution).     

Ultrastructure of Kurloff body proteoglycans after high pressure freezing, cryosubstitution and postembedding staining with cuprolinic blue

Very high pressure freezing and cryosubstitutlon of Kurloffcells preserves the ultrastructural morphology of Kurloff bodies,particularly the myelin figures, as shown by embedding in epoxyresin and conventional postembedding staining. It also preservesthe Kurloff body proteoglycans as more expanded spindle-likeshapes than does fixation with formaldehyde at atmospheric pressure.But., proteoglycans were not discernible in the Kurloff bodymatrix on either unstained or conventionally stained thin sections.The Kurloff body skeleton of proteoglycans in their native expandedshape was stained with the electron-dense cationic ministaincuprolinic blue, using thin sections embedded in LR white. Themean equatorial diameter of the spindles was 20–30 nm,while the collapsed filaments produced by aldehyde fixationwere about 10–15 nm wide. The spindles were often about200–300 nm long but could be much longer, depending onthe plane of the section. Thus, high pressure freezing, freezesubstitution, embedding in LR white, and staining with cationicdyes such as phthalocyanins seems to be a convenient way ofvisualizing intracellular proteoglycans that are well preservedand in very much like their native expanded state. cuprolinic blue high pressure freezing Kurloff cell proteoglycans ultrastructure     

Meta分析及其在生态学上的应用

１Ｍｅｔａ分析的概念和它的发展应用Ｍｅｔａ分析（Ｍｅｔａ－Ａｎａｌｙｓｉｓ）是一种定量综合研究结果的方法．这种方法的思想起源于本世纪３０年代,６０年代用于教育、心理等社会科学领域,１９７６年由Ｇｌａｓ命名为术语“Ｍｅｔａ－Ａｎａｌｙｓｉｓ”［１］。Ｇ...     

生态学中的干扰理论与应用实例

生态学中的干扰理论与应用实例魏斌张霞吴热风（北京大学环境科学中心,１００８７１）ＴｈｅｏｒｅｔｉｃａｌＡｎａｌｙｓｉｓｏｆＤｉｓｔｕｒｂａｎｃｅｉｎＥｃｏｌｏｇｙａｎｄｉｔｓＡｐｐｌｉｃａｔｉｏｎ．ＷｅｉＢｉｎ,ＺｈａｎｇＸｉａａｎｄＷｕＲｅｆｅｎｇ...     

Application of Solcograft in vascular surgery.

Incorporation of Solcograft was studied experimentally and clinically. The graft was found to facilitate the formation of continuous endothelium which starts at the vessel graft boundary and from newly formed capillaries. Transmission and scanning microscopical investigations revealed neointima, neomedia and neoadventitia formation in 3--4 months. Complications such as obliteration were unfrequent. Aneurysm formation did not occur, the potency rate was satisfacory. Experience in 20 clinical cases are reported in detail. As to its usefulness in reconstructive vascular surgery, Solcograft is placed behind the saphenous graft. The graft is particularly suitable for patch plastics.     

Application of theoretical models in ecology.

Certain general prey-predator models are compared with a particular ecosystem simulation. The adequacy of the models is considered in relation to the structure of the equations; to the representation of ecological features by terms in these equations; and to the need to incorporate resource equations in such models.     

稻曲病菌AFLP反应体系的建立及其初步应用

本研究通过一系列梯度试验建立了最佳的稻曲病菌AFLP(Amplifiedfragmentlengthpolymor-phism)反应体系,并从256对EcoRⅠ和MseⅠ引物组合中选择30对条带清晰、多态性好的引物组合分析了2003年北京昌平基地的40个稻曲病菌株的遗传多样性。     

Application of laser optical tweezers in immunology and molecular genetics.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.     

鹿蹄草植物的研究与应用

对鹿蹄草植物的自然资源分布、化学成分、药理作用、及其应用的相关研究做了综述。该植物在世界各地广泛分布、含有鹿蹄草素等多种化学成分,药理研究证明其具有抗菌、抗病毒、消炎、镇痛作用、对心脑血管系统等作用,主要应用在医药工业、食品工业和园林绿化面。最后对该属植物的研究前景做了展望。     

Application of topological methods in enzyme kinetics.

A criticism [Cornish-Bowden (1976) Biochem. J. 159, 167] of an algebraic method for deriving steady-state rate equations [Indge & Childs (1976) Biochem. J. 155, 567-570] is theoretically founded.     

Application of reversibly soluble polymers in bioprocessing.

Reversibly soluble polyelectrolytes, whose solubility in aqueous solutions is dependent on factors such as pH or ionic strength, may be used as supports for enzymes or affinity ligands. Their dual nature may be exploited to take advantage of the soluble form during, for example, enzymatic reactions, and the insoluble form during downstream processing operations for enzyme or product recovery.