Monoclonal antibodies against tomato spotted wilt virus: Characterisation and application*

Mouse hybridoma cells, secreting monoclonal antibodies (MCA) against tomato spotted wilt virus, were produced and screened for virus specificity by an indirect triple antibody ELISA, using a rabbit polyclonal antiserum for antigen trapping. A Bulgarian virus isolate from tobacco was used for immunisation of mice and rabbits. One fusion eventually led to 10 stable hybridoma cell lines, all of which produced antibodies of IgG-type though of different subgroups. Since none of the MCAs reacted with TSWV structural proteins after electrophoresis and transfer to nitrocellulose, other methods were chosen to examine their protein specificity. Purified viral cores and detergent-solubilised envelope proteins were used as antigens for ELISA, or, alternatively, glycosylated viral envelope proteins were trapped onto microtitre plates coated with lectins in order to detect MCAs specific for them. Both methods, independently, led to the identification of two MCAs that were specific for envelope proteins of TSWV. Only these two antibodies reacted with intact TSWV particles when examined by immunogold labelling in the electron microscope. The reaction of all MCAs with 11 different TSWV isolates eventually led to the selection of one core- and one envelope-specific antibody for routine use. Core-specific MCAs revealed serological differences between isolates belonging to the common serotype (= lettuce serotype), but did not react with the serotype TSWV-I. When comparing different ELISA procedures, broadest reactivity and highest sensitivity with different isolates were obtained in an indirect test procedure, using goat anti-mouse antibody conjugates.     

Scanning Electron Microscope Study of the Root-knot Nematode (Meloidogyne incognita) on Tomato Root

This study examines the types of structural information that can be gained by utilizing the scanning electron microscope (SEM) and a cryofracture technique to examine the host-parasite interaction. Roots of tomato, Lycopersicon esculentum cv. Marglobe, were cultured aseptically and inoculated with the root-knot nematode, Meloidogyne incognita. Twenty-four hours to four weeks after inoculation, developing galls were removed from the cultures and processed for SEM observation. The cryofracture technique was used to reveal internal structural features within the developing galls. The results illustrate structural details concerning penetration of the roots, differentiation of syncytia, and development of the nematodes beginning with the second-stage larvae and ending with adult egg-laying females.     

A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Antenna ring around trimeric Photosystem I in chlorophyll b containing cyanobacterium Prochlorothrix hollandica

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called “prochlorophytes”) that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

A scanning electron microscopic study of germ cell maturation in the reproductive tract of the male soupfin shark (Galeorhinus galeus)

Germ cell maturation in the reproductive tract of the soupfin shark (Galeorhinus galeus) was studied using scanning electron microscopy (SEM). The SEM showed changes in Sertoli cytoplasm volume during spermatogenic development. In immature spermatocysts in the germinal zone, spermatogonia were embedded in Sertoli cytoplasm. In spermatogonial spermatocysts, Sertoli cells were adluminally located in the spermatocyst, with spermatogonia enveloped in the basal portions of the cytoplasm. During the round spermatid stage, Sertoli cytoplasm was very scanty. Spermatid elongation was accompanied by a progressive increase in the volume of Sertoli cytoplasm, notably around the spermatid heads. In the mature spermatocyst, bundles of spermatozoa are totally enveloped by Sertoli cytoplasm. Spermatozoa occurred randomly in the epididymis. However, in the ampulla ductus deferentis, spermatozoa reaggregated and were embedded in a mucoid substance to form highly ordered spherical bundles. In the sperm bundle, the spermatozoa heads were arranged such that the helical turns of adjacent spermatozoa were precisely aligned, and all the heads in the bundle formed a distinct apex. This study demonstrates the utility of exploring the relationship between germ cells and Sertoli cells in an evolutionarily ancient vertebrate, such as the shark.     

Enhanced dark field microscopy for rapid artifact-free detection of nanoparticle binding to Candida albicans cells and hyphae

We surveyed a panel of 13 metal nanoparticle (NP) catalysts for their antifungal activities against Candida albicans ATCC 90028. Initial characterization using scanning electron microscopy (SEM) suggested that our ability to detect NP binding to Candida surfaces with this method was impeded by preparation artifacts. As an alternative method for visualizing NP binding, we used an enhanced dark field illumination system (CytoViva®) attached to a standard light microscope. When viewed using this system, all NP produced intense optical signals due to resonant light scattering. To assay binding, NP were allowed to interact with C. albicans hyphae and cells in spent RPMI broth for 15 min with gentle inversion, followed by viewing with the CytoViva® system. The antifungal efficacy of NP preparations was determined separately using a 24-h broth microdilution test. For single-metal NP, observations of binding at 15 min made via CytoViva® corresponded to antifungal efficacy at 24 h, with the most antifungal NP yielding complete coverage of hyphal surfaces. Our work suggests the utility of visual screening using the CytoViva® system for rapid, simple and artifact-free viewing of NP-cell interactions in support of antimicrobial screening efforts. This approach provides a quick and accessible alternative to SEM for imaging of NP-cell interactions.     

用感染病毒细胞超微结构的差异区分RMV及TMV株系

通过透射电镜观察被长叶车前草花叶病毒(RMV)和烟草花叶病毒(TMV)不同株系感染的普通烟叶肉细胞的超微结构,发现两种病毒的粒子分布、内含体类型、被感染细胞超微结构的病变均存在差异.病毒粒子分布有成束、分散、环状、膜包被及角状成层或平行成层排列等类型,存在于细胞质及液泡中,但未见于细胞核、线粒体及叶绿体等细胞器中.内含体的X-小管形状有长杆状、短杆状及颗粒状,数量各异.细胞壁常引起增厚、结构松散及扭曲等变化.叶绿体聚集成堆或分布于细胞边缘,其数量、大小、形状及所含淀粉粒、嗜锇颗粒等存在差异,有些还有颗粒状物质积累.线粒体及内质网等在不同株系间也存在差异.本项研究表明,被感染细胞超微结构的差异可作为RMV和TMV株系区分的依据.     

Studies on nucleoli of maturing frog erythroblasts

Summary Frog erythroblasts were studied in summer animals with a very active as well as reduced erythropoiesis due to experimental hibernation, the latter being administered in order to get more information on the frequency of various nucleolar types in maturing cells. The results suggest that nucleoli with nucleolonemata are a transitional nucleolar type between compact and ringshaped nucleoli. Since micronucleoli represent final nucleolar maturation changes and compact nucleoli are present in most immature cells, the sequence of nucleolar changes based on the frequency of investigated nucleolar types is as follows: compact nucleolinucleoli with nucleolonemataringshaped nucleolimicronucleoli. The experimental hibernation produces a shift of nucleoli to less active and maturer nucleolar types in all stages of the erythroblastic maturation. In addition, the experimental hibernation produces the formation of ringshaped nucleoli in the first stages of the erythroblastic maturation which in summer animals usually contain compact nucleoli and/or nucleoli with distinct nucleolonemata.