In vivo homologous recombination intermediates of yeast mitochondrial DNA analyzed by electron microscopy

Summary To study the structure of in vivo mitochondrial DNA recombination intermediates in Saccharomyces cerevisiae, we used a deletion mutant of the wild type mitochondrial genome. The mtDNA of this petite is composed of a direct tandem repetition of an 4,600 pb monomer repeat unit with a unique HhaI restriction enzyme site per repeat. The structure of native mtDNA isolated from log phase cells, and mtDNA crosslinked in vivo with trioxsalen plus UVA irradiation, was studied by electron microscopy. Both populations contained crossed strand Holliday type recombination intermediates. Digestion of both non-crosslinked and crosslinked and mtDNA with the enzyme HhaI released X and H shaped structures composed of two monomers. Electron microscopic analysis revealed that these structures had pairs of equal length arms as required for homologous recombination intermediates and that junctions could occur at points along the entire monomer length. The percentage of recombining monomers in both non-crosslinked and trioxsalen crosslinked mtDNA was calculated by quantitative analysis of all the structures present in an HhaI digest. The relationship between these values and the apparent dispersive replication of mtDNA in density-shift experiments and mtDNA fragility during isolation is discussed.     

Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast

A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K ^– R ^– was subjected to the K₂ killer effect of Saccharomyces cerevisiae T206 K ⁺ R ⁺ in a liquid grape medium. The lethal effect of the K₂ mycoviral toxin was confirmed by methylene blue staining. Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface. Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.     

Sequential scanning electron microscopy of a growing plant meristem

Summary A two-step replica technique has been developed for sequential study of the epidermal cell pattern of a living plant by scanning electron microscopy. This method is nondestructive, allows periodic high resolution observation of the same developing tissue, and can precede use of any destructive technique, such as transmission electron microscopy. The replicas can be trimmed allowing observation of occluded surfaces, such as the areas between leaves, which are inaccessible in continuousin vivo studies. Here we study the developing leaf primordium ofGraptopetalum and discuss potential uses of the technique.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Observation of Protoplasts from the Conidia of Arthrinium Aureum by scanning electron microscopy

Protoplast formation from conidia of Arthrinium aureum was achieved with Lysing Enzyme L-2265 (Sigma Chemical) from Trichoderma harzianum. Scanning electron microscopy of conidia protoplasts showed the acquired spherical shape.     

Observation of Woronin bodies in Arthrinium aureum by scanning electron microscopy

Woronin bodies are cytoplasmic organelles of filamentous fungi that can be observed on one, or both sides of each septum. The goal of this paper is to illustrate the presence of them in hyphae of Arthrinium aureum by means of scanning electron microscopy and to show that they act as a safety plug to close septa pores in hypha. Results show that Woronin bodies as an immediate response to prevent a cytoplasm loss. Results support hypothesis proposed previously in literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

The three-dimensional morphology of <Emphasis Type="Italic">Candida albicans</Emphasis> as seen by high-resolution scanning electron microscopy

The fine structure of Candida albicans has been repeatedly described by transmission electron microscopy, whereas studies by high-resolution scanning electron microscopy (HRSEM) are rare and devoted solely to the study of its external morphology. This report describes the results of an HRSEM study on C. albicans carried out by an osmium maceration protocol modified to better retain the structural characteristics of this yeast. Thus, we visualized various intracellular structures including invaginations of cell membrane (plasmalemmasomes), nuclear envelope, mitochondria, the vacuolar system, and two additional structures that might represent a form of endoplasmic reticulum and the Golgi apparatus. The present investigation, which for the first time shows the organelles of C. albicans at the 3D level, may lead to a better understanding of its cell physiology.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

Aspects of the three-dimensional intracellular organization of mesocarp cells as revealed by scanning electron microscopy

Summary The osmium-ligand binding technique and scanning electron microscopy have been applied to the study of the three-dimensional organization of mesocarp cells of a mature avocado fruit. Using this approach the mitochondria of the cells appear as elongated, branching structures and the endoplasmic reticulum consists of a complex of tubular strands, vesiculated strands and lamellar sheets. Associations of the endoplasmic reticulum with other organelles are also apparent. It is suggested that this approach provides a valuable means to assess the structural transitions in cell organization that occur during development or with functional changes.     

三疣梭子蟹精卵相互作用的扫描电镜观察

应用扫描电镜技术观察了三疣梭子蟹的精卵相互作用。未受精成熟卵表面较光滑、无受精孔,但有许多微孔。成熟卵外被卵膜,内为卵母细胞。在卵自然产出后,精子迅速发生顶体反应使顶体囊外翻并压入卵膜,而核仍留于卵膜外,核辐射臂不收缩且仍附着于卵膜上。三疣梭子蟹为多精着卵和多精入卵膜。精子外翻顶体囊压入卵膜后,核辐射臂陆续回缩直至消失。作用于顶体丝上的卵母细胞主动拖精作用对入卵膜精子的进一步入卵、受精至关重要,环状卵膜突起的向心伸展也有一定的协助作用。探讨了着卵精子的顶体反应、精子入卵膜的机制及卵子在精子入卵过程中的作用     

Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast

Reevaluation and comparison of seemingly contradictory literature data on the mode of synthesis of wall polysaccharides during the cell cycle ofSaccharomyces cerevisiae explained the source of discrepancies and demonstrated their general consonance in the following points: 1. The rate of synthesis of glucan and mannan is not constant and does not increase continuously throughout the entire cell cycle. 2. The rate of synthesis of both polysaccharides is considerably reduced at the time of cell division and in the prebudding phase.     

Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

Antifungal activity of allylamines on Epidermophyton floccosum: scanning electron microscopy study

The action of allylamine antifungal agents on Epidermophyton floccosum was studied using scanning electron microscopy. After 7 days of culture on Sabouraud dextrose agar, Epidermophyton floccosum samples were brought in contact with concentrations of 0.2 and 2 g ml^–1 and 0.01 and 0.1 g ml^–1 of naftifine and terbinafine, respectively. Lesions observed after 24 h, 3 and 7 days of contact were mainly on the structure and rigidity of the mycelial and macroconidial wall. They were characterized by hyphal ballooning and twisting and by apical bulbous bulges. Deterioration of macroconidia was characterized by wall exfoliation. The intensity of the deterioration depended on the dose and only slightly on the length of time that the sample and the antifungal drug were in contact.     

Scanning electron microscopy of the bullfrog's retina and pigment epithelium

Summary The retina and pigment epithelium of the bullfrog (Rana catesbiana) were studied with the scanning electron microscope. Fixed-dehydrated tissues were critical point dried with CO₂, then cracked in the plane of the long axis of the photoreceptors. The cellular layers of the retina and the lateral surfaces of pigment epithelial cells were visualized. The four major types of frog photoreceptor were identified: red rod, green rod, single cone, and double cone. Cone myoids were observed to be contracted in light-adapted retinas and elongated in more dark adapted retinas.This work was supported by a career development award EY-18,083 to the author and research grant EY 00468 to Dr. Kenneth T. Brown.The author gratefully acknowledges the skillful technical assistance of Ms. Maria T. Maglio.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

A critical reevaluation of the structure of the rat uriniferous tubule as revealed by scanning electron microscopy

Summary The fine structure of luminal surface of clearly identified portions of uriniferous tubules has been studied by scanning electron microscopy to elucidate some controversies concerning the topography of certain surface formations. The results show a characteristic pattern of the luminal surface in the region of Henle's loop, which was assumed by previous authors, to belong to the collecting tubule. Furthermore it is demonstrated that no cilia are present within the terminal portion of the collecting tubules.     

培养破骨细胞扫描电镜样本制备方法的探讨

目的：探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法：实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果：破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论：牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

培养破骨细胞扫描电镜样本制备方法的探讨

目的:探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法:实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果:破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论:牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。