Ultrastructure of the Ameboid Cells of Protostelium zonatum (Mycetozoa)*

SYNOPSIS. The ameboid cells of Protostelium zonatum typically have filose pseudopodia that contain almost exclusively microfibers. Microfibers, scattered or arranged parallel in bundles, are observed in great abundance and often closely associated or directly connected with other organelles in the cytoplasm. Mitochondria typically have an expanded, undulate outer membrane and vesicular cristae containing electron-dense material which may appear denser than the mitochondrial matrix in the same section. The nucleus, as in other protostelids, contains only one centrally located nucleolus. Other organelles are essentially similar in ultrastructure to those of another nonflagellate protostelid, Protosteliopsis fimicola. Protostelium zonatum differs, however, in lacking a distinct anterior zone of hyaloplasm during migration of the ameboid cells.     

Calcium-Induced Ultrastructural Interactions in Adrenocorticocytes

Steroidogenesis in adrenocorticocytes is closely related to intracellular [Ca²⁺]. To detect ultrastructural changes induced by growth in cytosolic [Ca²⁺], we used a rat adrenocortical cell culture, which was examined with electron microscopy and morphometric analysis. We established that either KCl-induced membrane depolarization evoking Ca²⁺ influx into the cell via voltage-operated Ca channels and Ca²⁺ ionophore, A23187, induced remarkable ultrastructural interactions between several cytosolic organelles. Lipid droplets known as key elements for Ca²⁺-induced steroidogenesis directly contacted with organelles containing the enzymes providing steroidogenic reactions (mitochondria, smooth and rough endoplasmic reticulum, nucleus, peroxisomes, and lysosomes). In most cases, the lipid droplets formed a specialized morphological structure at the sites of contact with the partner organelles. These structures are interpreted as a specialized transporting system, which provides contacts between organelles and exchange of intermediate products of the steroidogenesis process between the droplet and organelles.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

Comparative Studies of Calcium Transients Induced by Acetylcholine in Rat Chromaffin Cells

We used an intracellular fluorescent probe, FURA-2M, to examine the responses of isolated rat chromaffin cells to applications of 1 mM acetylcholine (ACh). Our data showed two different populations of the cell responses to such stimulation. Responses of the first type demonstrated fast rise and decay phases of the Ca²⁺ transients and no significant decrease in their amplitude during repetitive stimulation of the cell with ACh. Cell responses of the second type showed remarkably slower rise and decay phases of the Ca²⁺ transients and a noticeable drop of the cell responses during repetitive ACh stimulation that could be recovered after KCl depolarization. We find no significant differences in the amplitudes of the transients in these two populations of the cells. We conclude that there is heterogeneity of the chromaffin cells according to their ACh receptors: the first subpopulation predominantly expresses ionotropic (nicotinic) receptors (n cells), whereas the second cell population has mainly metabotropic (muscarinic) ones (m cells), which are associated with Ca²⁺ release from the intracellular stores.     

Circadian Adrenocortical Periodicity Changes in Adult Rats Stimulated During Early Development—II. Effect of Cold Stress

Rats were cooled at different time points of early postnatal development to study the effects of short-term stress on the parameters of the circadian rhythm of the adrenocortical function in adults. It was found that rats stressed at the age of 2-4 or 17-19 days exhibit modified tuning in the circadian periodicity of the adrenocortical function as adults. Cold stimulation during early development does not affect the function of isolated adrenal glands in vitro. It is concluded that the effect of early stress on the adrenocortical rhythmicity in adults is mediated through central regulatory mechanisms. This effect depends on the age when the rat pups are exposed to cold stress.     

Structural Changes in Tetrahymena rostrata during Induced Encystment*

SYNOPSIS. Experimentally induced precystic stages and mature cysts from 3 clones of Tetrahymena rostrata were examined by light and electron microscopy. It was demonstrated by cytochemical staining and fine-structural observations that precystic stages release mucocyst material that provides for the production of a cyst wall. Early and late cysts also contain numerous autophagous vacuoles. In late cysts there is a replacement of depleted mucocyst organelles. The developmental evidence obtained from sampling of sequential developmental stages suggests an ～24-h timetable of cytoplasmic events associated with encystment in this organism.     

Changes in Ultrastructure of Phaseolus Vulgaris L. Cotyledons Associated with their Modulated Life Span

French bean (Phaseolus vulgaris L.) cotyledons lost most of their reserve substances during several early days of germination and turned green. In cotyledon mesophyll cells of one-week-old seedlings, plastids were represented predominantly by amyloplasts (starch grains) and chloroamyloplasts, and the cells appeared to be metabolically highly active. Cell heterogeneity associated with distance of the cells from cotyledon vascular bundles was evident. Only mesophyll cells near to the bundles were rich in plastids. In two-weeks-old intact bean plants, the cotyledons were yellow and shrunken, and their cells were nearly "empty". The plastids in them were represented by senescent plastids (gerontoplasts) only. In the gerontoplasts as well as freely in cytosol, fluorescent lipoid inclusions were accumulated. This cotyledon development was more or less independent of irradiance. In "decapitated" bean plants, senescence of mesophyll cells and plastids was slowed down considerably, and the life span of the cotyledons was prolonged.     

纤毛虫念珠异列虫射出胞器的超微结构观察

应用扫描电镜术和透射电镜术显示,纤毛虫念珠异列虫(Anteholosticha monilata)的射出胞器早期发生在细胞质深处,附近有不同类型的囊泡结构。成熟后射出胞器向表膜迁移,结构由不同电子密度片层的体部、结晶状的中心轴杆部和多层膜的帽部组成。受外界刺激时胞器冲破皮层射出,形态呈"蘑菇"状。据上述观察结果推测:该射出胞器具有防御作用,它可能起源于高尔基体活动产生的小泡;在亲缘关系较近的纤毛虫中,其射出胞器可能具有相似的分化特征。     

Ultrastructure of the Invasion of Eimeria magna Sporozoites into Cultured Cells*†‡

SYNOPSIS. Monolayers of bovine kidney cells were overlaid with Eimeria magna sporozoites and observed with phase-contrast optics until penetration of the cells by the parasites had begun. Cells and penetrating parasites were fixed with glutaraldehyde and OsO₄-containing ruthenium red, dehydrated, and embedded in situ. Cells being penetrated were selected for study in the electron microscope. The lack of intracellular staining with ruthenium red and intact plasmalemmas of cells being penetrated, was accepted as evidence that the sporozoites did not disrupt the plasma membranes. The sporozoite caused invagination of the host cell plasmalemma until the parasite was entirely within the cell, after which the invagination was sealed off by short pseudopodia enclosing the sporozoite within a membrane-lined vacuole inside the cell. Often myelin-forms, apparently of host cell origin, were seen in the space between the sporozoite and the cell.     

A Simple Procedure for in Situ Immunolabeling, Embedding and Sectioning of Layers of Cultured Endothelial and Smooth Muscle Cells for both Light and Electron Microscopy

We present a simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy. Endothelial and smooth muscle cells were seeded in tissue culture chambers /slides precoated with 30% (w/v) gelatin drops fixed with 0.5% glutaraldehyde. Live endothelial cell layers were labeled with an antibody against the surface membrane protein, anti-CD 13. After labeling, the cell layers were fixed and separated from the chambers/slides by lifting all of the samples with a spatula. Sections (1-2 mm) were cut, embedded and processed further for light or electron microscopy. Because of the delicate cell layers and the importance of preserving maximum integrity, labeling was performed under standard culture conditions and treated in situ during the entire procedure. Moreover, the small chamber size of the tissue culture dishes generated the additional advantages of requiring only a limited number of cells, small volumes of media, and little antibody.     

Ultrastructure of the pre-implantation shark yolk sac placenta

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.     

Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor

Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ～30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [³⁵S]sulfate during the last 48 h of the culture were 1.5-to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [³⁵S]sulfate-labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [³⁵S]sulfate by PC12 cells was lower in the NGF-treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.     

Submicroscopic Changes in Visual Cells of the Rabbit Induced by Iodoacetate

Alterations produced by iodoacetate in visual cells have been studied under the electron microscope. Lesions of the outer segments of the rods are visible as early as 3 hours after a single injection of 20 mg. iodoacetate per kg. body weight. After 6 hours the changes are more marked and consist then of disorganization, vesiculation, and lysis of the rod sacs. The inner segments of most rod cells show swelling and vacuolization of the matrix, the endoplasmic reticulum, and the Golgi complex. The mitochondria of the ellipsoid show a tendency to disintegrate. In some inner segments the changes consist primarily in an increase in density of the matrix and deposition of a granular material. The rod synapses are also affected, showing lysis of the synaptic vesicles and alterations of the synaptic membrane. With a second injection of 20 mg. iodoacetate per kg. body weight, all these changes become more marked and lead to complete destruction of the rod cells. The cones seem more resistant than the rods. A single injection produces no visible changes in the outer or inner segments of the cones. At cone synapses, however, there are changes consisting of fusion of synaptic vesicles and other membranous material to form large concentric membranes characteristic of myelin figures. A second dose of the drug causes complete destruction of the cone cells. All these, and other submicroscopic changes, are discussed in relation to various hypotheses put forward to explain the mode of action of iodoacetate on visual cells. The pronounced alterations of submicroscopic intracellular membranes suggest that the locus of action of iodoacetate may be a component widely dispersed throughout the visual cells and related, in some way, to the maintenance of these lipoprotein structures.     

隐球菌脑膜炎患者治疗后隐球菌超微结构观察

目的研究隐球菌脑膜炎治疗后菌体超微结构的变化,探讨电镜检查在隐球菌活力检测中的应用。方法对8例经过两性霉素B和5-氟胞嘧啶联合治疗8周后,脑脊液中仍然可以查见隐球菌的患者,采用透射电镜对其脑脊液中的隐球菌进行超微结构观察。结果隐球菌菌体结构都显示了明显的变异：菌体大小差异显著,菌体形态变化明显;荚膜结构紊乱,菌体内可见空洞状或多个巨大脂滴,部分菌体胞膜破损,胞浆溢出。结论隐球菌脑膜炎治疗后虽然脑脊液中还存在菌体,但是菌体的超微结构已经发生了重大变化,提示菌体活力降低或死亡。电镜检查可以作为隐球菌活力判定的一种有效手段,提高隐球菌脑膜炎疗效判定的准确性。     

Ultrastructure of the retinal pigment epithelium in the young masu salmon Oncorhynchus masou

The ultrastructure of retinal pigment epithelium (RPE) cells in the masu salmon Oncorhynchus masou fry was studied. The physiological state of the pigment was dependent on the level of light/dark adaptation. Similar morphological properties were observed in the response of the RPE cells to light and a magnetic field.     

低温下的绿豆下胚轴细胞超微结构变化

采用透射电子显微镜观察经10℃低温处理2d的绿豆下胚轴细胞超微结构变化的结果表明,细胞受到不同程度的伤害,其中大部分细胞的损伤是可逆的而非致死性的,如内质网膜模糊和呈颗粒状;线粒体呈现不同程度的膨胀,其数量增加,且聚集分布在内质网和高尔基体附近,核糖体聚集成为多聚核糖体等。也有小部分细胞受到的伤害是致死性的,如细胞质电子密度显著升高,质体中淀粉粒过度积累,内质网和高尔基体膨胀和解体,小液泡频繁吞噬细胞质和细胞器,细胞自溶死亡等。     

The Ultrastructure of Chlorobium tepidum Chlorosomes Revealed by Electron Microscopy

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.     

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission election microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.